Two‐Photon Absorption and Time‐Resolved Stimulated Emission Depletion Spectroscopy of a New Fluorenyl Derivative

The synthesis, comprehensive linear photophysical characterization, two‐photon absorption (2PA), steady‐state and time‐resolved stimulated emission depletion properties of a new fluorene derivative, (E)‐1‐(2‐(di‐p‐tolylamino)‐9,9‐diethyl‐9H‐fluoren‐7‐yl)‐3‐(thiophen‐2‐yl)prop‐2‐en‐1‐one ( 1 ), are reported. The primary linear spectral properties, including excitation anisotropy, fluorescence lifetimes, and photostability, were investigated in a number of aprotic solvents at room temperature. The degenerate 2PA spectra of 1 were obtained with open‐aperture Z‐scan and two‐photon induced fluorescence methods, using a 1 kHz femtosecond laser system, and maximum 2PA cross‐sections of ～400–600 GM were obtained. The nature of the electronic absorption processes in 1 was investigated by DFT‐based quantum chemical methods implemented in the Gaussian 09 program. The one‐ and two‐photon stimulated emission spectra of 1 were measured over a broad spectral range using a femtosecond pump–probe‐based fluorescence quenching technique, while a new methodology for time‐resolved fluorescence emission spectroscopy is proposed. An effective application of 1 in fluorescence bioimaging was demonstrated by means of one‐ and two‐photon fluorescence microscopy images of HCT 116 cells containing dye encapsulated micelles.     

Excited State Intramolecular Proton Transfer and Photophysics of a New Fluorenyl Two‐Photon Fluorescent Probe

The steady‐state photophysical, NMR, and two‐photon absorption (2PA) properties of a new fluorene derivative ( 1 ) containing the 2‐(2′‐hydroxyphenyl)benzothiazole (HBT) terminal construct is investigated for use as a fluorescence probe in bioimaging. A comprehensive analysis of the linear spectral properties reveals inter‐ and intramolecular hydrogen bonding and excited state intramolecular proton transfer (ESIPT) processes in the HBT substituent. A specific electronic model with a double minimum potential energy surface is consistent with the observed spectral properties. The 2PA spectra are obtained using a standard two‐photon induced fluorescence method with a femtosecond kHz laser system, affording a maximum 2PA cross section of ～600 GM, a sufficiently high value for two‐photon fluorescence imaging. No dependence of two‐photon absorption efficiency on solvent properties and hydrogen bonding in the HBT substituent is observed. The potential use of this fluorenyl probe in bioimaging is demonstrated via one‐ and two‐photon fluorescence imaging of COS‐7 cells.     

Superfluorescent Squaraine with Efficient Two‐Photon Absorption and High Photostability

The synthesis, linear photophysical, two‐photon absorption (2PA), femtosecond transient absorption, and superfluorescence properties of a new symmetrical squaraine derivative ( 1 ) are reported. Steady‐state linear spectral and photochemical properties, fluorescence lifetimes, and excitation anisotropy of 1 were investigated in various organic solvents. High fluorescence quantum yields (≈0.7) and very high photostability (photodecomposition quantum yields ≈10^?6–10^?8) were observed. An open‐aperture Z‐scan method was used to obtain 2PA spectra of 1 over a broad spectral range (maximum 2PA cross section ≈1000 GM). Excited‐state absorption (ESA) and gain was observed by femtosecond transient absorption spectroscopy, in which both reached a maximum at approximately 500 fs. Squaraine 1 exhibits efficient superfluorescence. The quantum chemical study of 1 revealed the simulated vibronic nature of the 1PA and 2PA spectra were in good agreement with experimental data; this may provide the ability to predict potential advanced photonic materials.     

Luminescent Quadrupolar Borazine Oligomers: Synthesis,Photophysics, and Two‐Photon Absorption Properties

A set of monodisperse bent donor–acceptor–donor‐type conjugated borazine oligomers, BnNn+1 (n=1–4), incorporating electron‐rich triarylamine donor and electron‐deficient triarylborane acceptor units has been prepared through an iterative synthetic approach that takes advantage of highly selective silicon–boron and tin–boron exchange reactions. The effect of chain elongation on the electrochemical, one‐ and two‐photon properties and excited‐state photodynamics has been investigated. Strong intramolecular charge transfer (ICT) from the arylamine donors to boryl‐centered acceptor sites results in emissions with high quantum yields (Φ_fl>0.5) in the range of 400–500 nm. Solvatochromic effects lead to solvent shifts as large as ～70 nm for the shortest member (n=1) and gradually decrease with chain elongation. The oligomers exhibit strong two‐photon absorption (2PA) in the visible spectral region with 2PA cross sections as large as 1410 GM (n=4), and broadband excited‐state absorption (ESA) attributed to long‐lived singlet–singlet and radical cation/anion absorption. The excited‐state dynamics also show sensitivity to the solvent environment. Electrochemical observations and DFT calculations (B3LYP/6‐31G*) reveal spatially separated HOMO and LUMO levels resulting in highly fluorescent oligomers with strong ICT character. The BnNn+1 oligomers have been used to demonstrate the detection of cyanide anions with association constants of log K>7.     

Two‐Photon Polarity Probes Built from Octupolar Fluorophores: Synthesis,Structure–Properties Relationships,and Use in Cellular Imaging

A series of octupolar fluorophores built from a triphenylamine (TPA) core connected to electron‐withdrawing (EW) peripheral groups through conjugated spacers has been synthesized. Their photoluminescence, solvatochromism, and two‐photon absorption (2PA) properties were systematically investigated to derive structure–property relationships. All derivatives exhibit two 2PA bands in the 700–1000 nm region: a first band at low energy correlated with a core‐to‐periphery intramolecular charge transfer that leads to an intense 1PA in the blue‐visible range, and a second more intense band at higher energy due to an efficient coupling of the branches through the TPA core. Increasing the strength of the EW end groups or the length of the conjugated spacers and replacing triple‐bond linkers with double bonds induces both enhancement and broadening of the 2PA responses, thereby leading to cross‐sections up to 2100 GM at peak and higher than 1000 GM over the whole 700–900 nm range. All derivatives exhibit intense photoluminescence (PL) in low‐ to medium‐polarity environments (with quantum yields in the 0.5–0.9 range) and display a strong positive solvatochromic behavior (with Lippert–Mataga specific shifts ranging from 15 000 to 27 500 cm^?1), triple bonds, and phenyl moieties in the conjugated spacers, thereby leading to larger sensitivities than those of double bonds and thienyl moieties. More hydrophilic derivatives were also shown to be biocompatible, to retain their 2PA and PL properties in biological conditions, and finally to be suitable as polarity sensors for multiphoton cell imaging.     

Polar Diketopyrrolopyrrole‐Imidazolium Salts as Selective Probes for Staining Mitochondria in Two‐Photon Fluorescence Microscopy

Three rationally designed polar derivatives of diketopyrrolopyrrole consisting of 1,3‐dimethylimidazolium cationic units and benzene, thiophene, or furan rings as π spacers were synthesized and thoroughly studied. The obtained salts are soluble in polar organic solvents and show satisfactory solubility in water, which makes them suitable for the applications in bioimaging. Photophysical measurements revealed that the obtained derivatives are characterized by strong absorption and good fluorescence quantum yields. The corresponding two‐photon properties were also examined and showed that the synthesized salts exhibit large two‐photon absorption cross‐sections reaching 4000 GM (GM=Goeppert‐Mayer unit, 1 GM=10^?50 cm⁴ s photon^?1) and very high two‐photon brightness values exceeding 2000 GM. It was demonstrated that these salts can be safely applied in two‐photon fluorescence microscopy for selective staining of mitochondria in living cells.     

Water‐Soluble Triarylborane Chromophores for One‐ and Two‐Photon Excited Fluorescence Imaging of Mitochondria in Cells

Three water‐soluble tetracationic quadrupolar chromophores comprising two three‐coordinate boron π‐acceptor groups bridged by thiophene‐containing moieties were synthesised for biological imaging applications. Compound 3 containing the bulkier 5‐(3,5‐Me₂C₆H₂)‐2,2′‐(C₄H₂S)₂‐5′‐(3,5‐Me₂C₆H₂) bridge is stable over a long period of time, exhibits a high fluorescence quantum yield and strong one‐ and two‐photon absorption (TPA), and has a TPA cross section of 268 GM at 800 nm in water. Confocal laser scanning fluorescence microscopy studies in live cells indicated localisation of the chromophore at the mitochondria; moreover, cytotoxicity measurements proved biocompatibility. Thus, chromophore 3 has excellent potential for one‐ and two‐photon‐excited fluorescence imaging of mitochondrial function in cells.     

Boron Difluoride Curcuminoid Fluorophores with Enhanced Two‐Photon Excited Fluorescence Emission and Versatile Living‐Cell Imaging Properties

The synthesis of boron difluoride complexes of a series of curcuminoid derivatives containing various donor end groups is described. Time‐dependent (TD)‐DFT calculations confirm the charge‐transfer character of the second lowest‐energy transition band and ascribe the lowest energy band to a “cyanine‐like” transition. Photophysical studies reveal that tuning the donor strength of the end groups allows covering a broad spectral range, from the visible to the NIR region, of the UV–visible absorption and fluorescence spectra. Two‐photon‐excited fluorescence and Z‐scan techniques prove that an increase in the donor strength or in the rigidity of the backbone results in a considerable increase in the two‐photon cross section, reaching 5000 GM, with predominant two‐photon absorption from the S₀–S₂ charge‐transfer transition. Direct comparisons with the hemicurcuminoid derivatives show that the two‐photon active band for the curcuminoid derivatives has the same intramolecular charge‐transfer character and therefore arises from a dipolar structure. Overall, this structure–relationship study allows the optimization of the two‐photon brightness (i.e., 400–900 GM) with one dye that emits in the NIR region of the spectrum. In addition, these dyes demonstrate high intracellular uptake efficiency in Cos7 cells with emission in the visible region, which is further improved by using porous silica nanoparticles as dye vehicles for the imaging of two mammalian carcinoma cells type based on NIR fluorescence emission.     

Alternating copolymers of 2,6‐bis(p‐dihexylaminostyryl)anthracene‐9,10‐diyl and N‐octylcarbazole with large two‐photon cross sections

We report the synthesis, thermal, one‐ and two‐photon properties of poly(2,6‐bis(p‐dihexylaminostyryl)anthracene‐9,10‐diyl‐alt‐N‐octylcarbazole‐3,6‐/2,7‐diyl) ( P1/P2 ). The as‐synthesized polymers exhibit number‐average molecular weights of 1.7 × 10⁴ for P1 and 2.1 × 10⁴ g/mol for P2 . They emit strong one‐ and two‐photon excitation fluorescence with the peak around 502 nm, and the fluorescence quantum yields around 0.76 in chloroform. In film state, P1 and P2 show different red‐shift emission with the peaks at 512 nm and 523 nm, respectively. The DSC measurement reveals that as‐synthesized polymers are all amorphous aggregates with the glass transition temperatures of 131 °C for P1 and 152 °C for P2 . The solution two‐photon absorption (TPA) properties of P1 and P2 in chloroform are measured by the two‐photon‐induced fluorescence method using femtosecond laser pulses (120 fs). The TPA cross sections (δ) are measured over the range of 700–900 nm. The maximal δ of P1 and P2 all appear at ～800 nm and are 1010 GM and 940 GM per repeating unit, respectively. This suggests that no notable interactions among structure units that impair their fluorescence and TPA properties, and the polymers with large δ can be obtained by using the high TPA‐active units as building blocks. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Synthesis,Photophysical Properties and Two‐Photon Absorption Study of Tetraazachrysene‐based N‐Heteroacenes

Three novel N‐heteroacene molecules ( SDNU‐1 , SDNU‐2 and SDNU‐3 ) based on tetraazachrysene units as cores have been designed, synthesized and fully characterized. Their photophysical, electrochemical and fluorescence properties were investigated, and they exhibited blue to green emission in the solid state. Interestingly, SDNU‐2 exhibited high solid photoluminescence quantum efficiencies (75.3 %), which is the highest value of N‐heteroacenes derivatives to date. Two‐photon absorption studies have been conducted by using the open and close aperture Z‐san technique. SDNU‐3 showed a significant enhancement in the two‐photon absorption cross‐section with magnitudes as high as about 700 GM (1 GM=1×10^?50 cm⁴ s/photon) when excited with 800 nm light, which is the largest value based on a heteroacene system measured by using a Z‐scan experiment so far. We attribute the outcome to sufficient electronic coupling between the strong charge transfer of quadrupolar substituents and the tetraazachrysene core. Our result would provide a new guideline to design novel efficient two‐photon materials based on N‐heteroacene cores.     

Synthesis and Characterization of Two‐Photon Active Chromophores Based on Tetrathienoacene (TTA) and Dithienothiophene (DTT)

Three new donor–π–donor (D‐π‐D) tetrathienoacene (thieno[2′,3′:4,5]thieno[3,2‐b]thieno[2,3‐d]thiophene (TTA))‐cored chromophores, end‐functionalized with electron‐donating triphenylamine (TPA) groups, were developed and characterized for their two‐photon‐related properties by using both nano‐ and femtosecond laser pulses as the probing tools. TTA‐based chromophores exhibit stronger and more widely dispersed two‐photon absorption (2PA) than those of dithienothiophene (DTT)‐based congeners. As a consequence, the bithiophene‐conjugated TTA chromophore exhibits the highest maximum 2PA cross‐section value (up to 2500 GM) with good thermal stability, and thus, it is the best performing two‐photon chromophore among the studied model compounds. The bithiophene‐conjugated DTT analogue exhibits the second highest maximum two‐photon absorptivity of 1950 GM, which is nearly 7 times larger than that of previously reported DTT‐based chromophores.     

Difluorenyl carbo‐Benzenes: Synthesis,Electronic Structure,and Two‐Photon Absorption Properties of Hydrocarbon Quadrupolar Chromophores

The synthesis, crystal and electronic structures, and one‐ and two‐photon absorption properties of two quadrupolar fluorenyl‐substituted tetraphenyl carbo‐benzenes are described. These all‐hydrocarbon chromophores, differing in the nature of the linkers between the fluorenyl substituents and the carbo‐benzene core (C?C bonds for 3 a , C?C?C?C expanders for 3 b ), exhibit quasi–superimposable one‐photon absorption (1PA) spectra but different two‐photon absorption (2PA) cross‐sections σ_2PA. Z‐scan measurements (under NIR femtosecond excitation) indeed showed that the C?C expansion results in an approximately twofold increase in the σ_2PA value, from 336 to 656 GM (1 GM=10^?50 cm⁴ s molecule^?1 photon^?1) at λ=800 nm. The first excited states of A_u and A_g symmetry accounting for 1PA and 2PA, respectively, were calculated at the TDDFT level of theory and used for sum‐over‐state estimations of σ_2PA(λ_i), in which λ_i=2 hc/E_i, h is Planck’s constant, c is the speed of light, and E_i is the energy of the 2PA‐allowed transition. The calculated σ_2PA values of 227 GM at 687 nm for 3 a and 349 GM at 708 nm for 3 b are in agreement with the Z‐scan results.     

Pyrrolo[3,2‐b]pyrroles—From Unprecedented Solvatofluorochromism to Two‐Photon Absorption

A combined experimental and theoretical study of the two‐photon absorption (2PA) properties of a series of quadrupolar molecules possessing a highly electron‐rich heterocyclic core, pyrrolo[3,2‐b]pyrrole, is presented. In agreement with quantum‐chemical calculations, large 2PA cross‐section values, σ_2PA≈10²–10³ GM (1 GM=10⁵⁰ cm⁴ s photon^?1), are observed at wavelengths of 650–700 nm, which correspond to the two‐photon allowed but one‐photon forbidden transitions. The calculations also predict that increased planarity of this molecule through removal of two N‐substituents leads to further increase in the σ_2PA values. Surprisingly, the most quadrupolar pyrrolo[3,2‐b]pyrrole derivative, containing two 4‐nitrophenyl substituents at positions 2 and 5, demonstrates a very strong solvatofluorochromic effect, with a fluorescence quantum yield as high as 0.96 in cyclohexane, whereas the fluorescence vanishes in DMSO.     

Glycosylated Star‐Shaped Conjugated Oligomers for Targeted Two‐Photon Fluorescence Imaging

A glucopyranose functionalized star‐shaped oligomer, N‐tris{4,4′,4′′‐[(1E)‐2‐(2‐{(E)‐2‐[4‐(benzo[d]thiazol‐2‐yl)phenyl]vinyl}‐9,9‐bis(6‐2‐amido‐2‐deoxy‐1‐thio‐β‐D ‐glucopyranose‐hexyl)‐9H‐fluoren‐7‐yl)vinyl]phenyl}phenylamine (TVFVBN‐S‐NH₂), is synthesized for two‐photon fluorescence imaging. In water, TVFVBN‐S‐NH₂ self‐assembles into nanoparticles with an average diameter of ～49 nm and shows a fluorescence quantum yield of 0.21. Two‐photon fluorescence measurements reveal that TVFVBN‐S‐NH₂ has a two‐photon absorption cross‐section of ～1100 GM at 780 nm in water. The active amine group on the glucopyranose moiety allows further functionalization of TVFVBN‐S‐NH₂ with folic acid to yield TVFVBN‐S‐NH₂FA with similar optical and physical properties as those for TVFVBN‐S‐NH₂. Cellular imaging studies reveal that TVFVBN‐S‐NH₂FA has increased uptake by MCF‐7 cells relative to that for TVFVBN‐S‐NH₂, due to specific interactions between folic acid and folate receptors on the MCF‐7 cell membrane. This study demonstrates the effectiveness of glycosylation as a molecular engineering strategy to yield water‐soluble materials with a large two‐photon absorption (TPA) cross‐section for targeted cancer‐cell imaging.     

Synthesis,Two‐Photon Absorption and Optical Limiting Properties of Multi‐branched Styryl Derivatives Based on 1,3,5‐Triazine

Five new multi‐branched two‐photon absorption triazine chromophores ( T1 – T5 ) with different donor strength, conjugation length, and direction of charge transfer have been designed and synthesized. The one‐photon fluorescence, fluorescence quantum yields, and two‐photon properties have been investigated. The two‐photon absorption (2PA) cross sections measured by the open aperture Z‐scan technique were determined to be 447, 854, 1023, 603, and 766 GM for T1 , T2 , T3 , T4 , and T5 , respectively. This result indicates that their 2PA cross section values (σ) increase with increasing electron‐donating strength of the end group, extending the conjugation length of the system, and introducing electron‐withdrawing perfluoroalkyl as side groups to the end donor. In addition, the σ value of T5 is also larger than that of T1 , which provides evidence that the σ value is relative to the direction of charge transfer (from the ends to the center of the molecule or from the center to the ends). Moreover, significant enhancement of the two‐photon absorption cross section was achieved by introducing a thiophene moiety to a conjugated CC bond. At the same time, the optical limiting behavior for these chromophores was studied by using a focused 800 nm laser beam with pulses of 140 fs duration. It was found that these molecules also exhibit good optical limiting properties. These initial results clearly demonstrate that multi‐branched triazine chromophores are a highly suitable class of two‐photon absorbing materials.     

Synthesis and two‐photon absorption properties of conjugated polymers with N‐arylpyrrole as conjugated bridge and isolation moieties

Three novel conjugated polymers with N‐arylpyrrole as the conjugated bridge were designed and synthesized, which emitted strong one‐ or two‐photon excitation fluorescence in dilute tetrahydrofuran (THF) solution with high quantum yields. The maximal two‐photon absorption (TPA) cross‐sections of the polymers, measured by the two‐photon‐induced fluorescence method using femtosecond laser pulses in THF, were 752, 1114, and 1869 GM, respectively, indicating that the insertion of electron‐donating or electron‐withdrawing moieties into the polymer backbone could benefit to the increase of the TPA cross‐section. Their large TPA cross‐sections, coupled with the relatively high emission quantum yields, made these conjugated polymers attractive for practical applications, especially two‐photon excited fluorescence. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Two‐Photon Absorption Cross‐Sections of Reference Dyes: A Critical Examination

The electronic structure and one‐ and two‐photon absorption spectra of four fluorophores, p‐bis(o‐methoxystyryl)benzene (Bis‐MSB), coumarin 307, fluorescein and rhodamine B, commonly used as reference compounds for two‐photon absorption spectra, have been theoretically calculated and compared with available experimental data. The possible reasons for the wide discrepancies in two‐photon absorption cross‐sections reported in the literature are discussed on the basis of the theoretical findings. The role of a solvent environment on the electronic one‐ and two‐photon absorption spectra is also studied. We highlight some necessary precautions that one needs to take when comparing literature results of two‐photon absorption cross‐sections.     

Evidence for Two‐Photon Absorption‐Induced ESIPT of Chromophores Containing Hydroxyl and Imino Groups

This paper presents experimental and theoretical investigations into excited‐state intramolecular proton transfer (ESIPT) in new chromophores with hydroxyl and imino groups under one‐ and two‐photon excitation. The results show that internal hydrogen bonding exhibits a remarkable influence on the maximum absorption wavelength of 2‐[(4′‐N,N‐diethylaminodiphenylethylene‐4‐ylimino)methyl]phenol ( C1 ) and 2‐[(4′‐methoxyl‐diphenylethylene‐4‐ylimino)methyl]phenol ( C3 ). Compounds C1 and C3 exhibit well‐separated dual fluorescence emission bands under one‐ and two‐photon excitation. The second fluorescence peaks of C1 and C3 are characterized by much larger Stokes shift than the first normal peaks (ca. 140 vs. 30 nm). 4‐[(4′‐N,N‐Diethylaminodiphenylethylene‐4‐ylimino)methyl]phenol ( C2 ) and 4‐[(4′‐methoxyldiphenylethylene‐4‐ylimino)methyl]phenol ( C4 ) display single emission bands with small Stokes shifts (ca. 30 nm) in various solvents under one‐ and two‐photon excitation. Furthermore, the first emission maxima of C1 and C3 are almost identical to the maximum fluorescence emission wavelengths of C2 and C4 , respectively. These results show that C1 and C3 can undergo ESIPT via a reasonable six‐membered ring, while there is no ESIPT in C2 and C4 under one‐ and two‐photon excitation. Compounds C1 and C2 have larger two‐photon absorption cross‐sections under various near‐infrared laser frequencies tuned from 700 to 880 nm. Molecular geometry optimization of the phototautomers (enol and keto) was performed to analyze the experimental results. The possibility of using these chromophores for metal ions as chemosensors of was thoroughly investigated. In DMF C3 exhibits excellent sensing responses to Zn²⁺ and Fe³⁺ ions through a greatly increased greatly and a largely reduced emission, respectively. In methanol disappearance of ESIPT emission with added Zn²⁺ ions confirms its existence. The binding constants of C3 with Zn²⁺ and Fe³⁺ ions in DMF are also estimated.     

Energy Transfer in Aminonaphthalimide‐Boron‐Dipyrromethene (BODIPY) Dyads upon One‐ and Two‐Photon Excitation: Applications for Cellular Imaging

Aminonaphthalimide–BODIPY energy transfer cassettes were found to show very fast (k_EET≈10¹⁰–10¹¹ s^?1) and efficient BODIPY fluorescence sensitization. This was observed upon one‐ and two‐photon excitation, which extends the application range of the investigated bichromophoric dyads in terms of accessible excitation wavelengths. In comparison with the direct excitation of the BODIPY chromophore, the two‐photon absorption cross‐section δ of the dyads is significantly incremented by the presence of the aminonaphthalimide donor [δ≈10 GM for the BODIPY versus 19–26 GM in the dyad at λ_exc=840 nm; 1 GM (Goeppert–Mayer unit)=10^?50 cm⁴ s molecule^?1 photon^?1]. The electronic decoupling of the donor and acceptor, which is a precondition for the energy transfer cassette concept, was demonstrated by time‐dependent density functional theory calculations. The applicability of the new probes in the one‐ and two‐photon excitation mode was demonstrated in a proof‐of‐principle approach in the fluorescence imaging of HeLa cells. To the best of our knowledge, this is the first demonstration of the merging of multiphoton excitation with the energy transfer cassette concept for a BODIPY‐containing dyad.     

Two‐Photon Probes for Zn2+ Ions with Various Dissociation Constants. Detection of Zn2+ Ions in Live Cells and Tissues by Two‐Photon Microscopy

A series of Zn²⁺‐selective two‐photon fluorescent probes (AZnM1−AZnN) that had a wide range of dissociation constants (K_d^TP=8 nm‐ 12 μM ) were synthesized. These probes showed appreciable water solubility (>3 μM ), cell permeability, high photostability, pH insensitivity at pH>7, significant two‐photon action cross‐sections (86–110 GM) upon complexation with Zn²⁺, and can detect the Zn²⁺ ions in HeLa cells and in living tissue slices of rat hippocampal at a depth of >80 μm without mistargeting and photobleaching problems. These probes can potentially find application in the detection of various amounts of Zn²⁺ ions in live cells and intact tissues.