Small-Molecule Two-Photon Probes for Bioimaging Applications

Review pubs.acs.org/CR

Small-Molecule Two-Photon Probes for Bioimaging Applications Hwan Myung Kim*,† and Bong Rae Cho*,‡ †

Department of Chemistry & Energy Systems Research, Ajou University, Suwon 443-749, Korea Department of Chemistry, Korea University, 145, Anam-ro, Seoul 136-713, Korea 1. INTRODUCTION

‡

Small-molecule fluorescent probes are attractive and versatile materials for studying biological systems.1−6 They can be easily loaded into cells and rapidly detect biological targets, which are important requirements for cell-imaging reagents. Moreover, the binding affinity, kinetics, excitation/emission wavelength, and localization to a specific organelle can easily be optimized by using well-established design strategies.1−6 Thus, a diverse array of probes can be designed to target various biomolecules and organelles.7,8 Most of these have been developed by employing common fluorophores, such as fluorescein, coumarin, BODIPY, and rhodamine.9 These are useful for one-photon microscopy (OPM), which utilizes one photon (OP) of UV− visible light (350−550 nm). To date, thousands of OP probes have been developed, of which about 2000 have been commercialized. However, the use of OPM is limited to cell imaging and is not useful for deep-tissue imaging due to the shallow depth of tissue penetration (less than 100 μm) of the short excitation light, the cellular absorption of the photons, and autofluorescence induced by exciting intrinsic fluorophores, such as flavin adenine dinucleotide and nicotinamide adenine dinucleotide.10 During the past decade, two-photon microscopy (TPM), which utilizes two near-infrared (NIR) photons as the excitation source, has emerged as a new, indispensable imaging tool for biomedical research.11−15 Compared with OPM, TPM offers several advantages, including the capability of imaging deep inside a tissue, higher spacial resolution, and longer observation time. However, most of the fluorescent probes employed for TPM are OP probes. Since they are not optimized for TPM, they have small TP absorption (TPA) cross sections (δmax < 50 GM) that limit their use in TPM.16,17 To facilitate the use of TPM in biomedical research, there is a strong need for the development of a variety of efficient, application-specific, molecular TP probes. Importantly, recent model studies have confirmed that TP probes can be designed using the same strategies utilized for OP probe design, with the exception that an efficient TP fluorophore should be used. Following this guideline, a variety of TP probes have been developed, and their utility in bioimaging applications has been demonstrated. In the current literature, there are a number of reviews published on the use of TP probe for specific targets, including metal ions, organelles, mitochondria-specific ions and molecules, and bioconjugates.18−23 However, this literature is widely dispersed through a number of academic areas, and there is currently no single comprehensive review on this rapidly

CONTENTS 1. 2. 3. 4. 5.

Introduction Two-Photon Absorption Model Studies on Two-Photon Probes Design of Two-Photon Probes Two-Photon Probes for Lysosomes and Mitochondria 6. Two-Photon Probes for Plasma Membranes 7. Two-Photon Probes for Metal Ions 7.1. Two-Photon Probes for Sodium Ions 7.2. Two-Photon Probes for Magnesium Ions 7.3. Two-Photon Probes for Calcium Ions 7.4. Two-Photon Probes for Copper Ions 7.5. Two-Photon Probes for Zinc Ions 7.6. Two-Photon Probes for Mercury, Cadmium, Lead, and Nickel Ions 8. Two-Photon Probes for pH 9. Two-Photon Probes for Thiols and Hydrogen Sulfide 10. Two-Photon Probes for Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) 11. Two-Photon Probes for Glucose and ATP 12. Two-Photon Probes for Nucleic Acids 13. Two-Photon Probes for Enzymes 14. Two-Photon Fluorescent Bioconjugates 15. Two-Photon Probes for Medical Applications 16. Conclusions and Outlook Author Information Corresponding Authors Notes Biographies Acknowledgments References

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Received: August 13, 2014 Published: May 4, 2015 © 2015 American Chemical Society

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Figure 1. Energy diagrams showing one-photon absorption (OPA), two-photon absorption (TPA), and fluorescence for a molecule (a) without an inversion center (dipole and octupole) or (b) with an inversion center (quadrupole). (c) Representative structural motifs of an electron donor− acceptor (D−A) substituted dipole, quadrapole (D−A−D), and two-dimensional octupole.

The possibility of TPA was theoretically predicted by GöppertMayer in 193124 and experimentally confirmed by Kaiser and Garrett in 1961 after the invention of high-energy pulsed lasers.25 TPA can be classified as degenerate or nondegenerate, depending upon whether the two photons have the same or different energies, respectively. In degenerate TPA, which is more useful for practical applications, the energy level of the TP-allowed state of a molecule with or without an inversion center (i.e., quadrupoles versus dipoles and octupoles, respectively) is similar to or higher than that of the OPallowed state (Figure 1).26−28 As such, the former can reach the excited state by absorbing either OP (one-photon absorption, OPA) with an energy (ΔE) equal to the difference between the ground (g) and excited state (e) or two photons with ΔE/2, while the latter requires either the energy required for OPA (ΔE′) or TPs with energy greater than ΔE′/2 to be excited (Figure 1b). An exception to this OPA/TPA energy model is long chain polyenes, for which the energy level of the TPallowed state can be lower than that of the OP-allowed state.29 A lower-energy photon has been shown to have a longer wavelength that can penetrate deeper into the sample compared to a higher-energy photon, thereby allowing TP excitation to occur deeper inside a light-scattering medium. In addition, the probability of TPA (N2) increases with the square of the light intensity (I); that is, N2 ∝ δI2, where δ is the TPA cross section in Göppert-Mayer units (1 GM = 10−50 cm4 s photon−1 molecule−1).26−28 Unlike OPA, the efficiency of

growing subject. We believe this may limit the use of previously developed TP probes and hinder the discovery of others, stifling overall growth in the field of TPM. Therefore, in the current review, we present a condensed, all-inclusive evaluation of the design, synthesis, and biomedical application of the various small-molecule TP probes that have been published prior to December 2014. In order to avoid an excessive number of references, we have focused on only the TP probes for which the properties have been clearly characterized and which produce high-quality TPM images. As a result, we apologize to the authors, particularly those currently synthesizing, characterizing, and fine-tuning the application of previously undocumented probes, whose results have not been included here. We begin by describing the fundamental concept of TPA, followed by a description of the organic solvent-based model studies that established the use of TPM in the biomedical field. Further, we then provide an overview of the basic strategies used to design a TP probe and highlight selected examples that have been used to detect biologically important targets in vivo. Our analysis is concluded with a discussion of the current utilization and performance of TP probes in various medical applications as well as the future outlook for this continually expanding area of research.

2. TWO-PHOTON ABSORPTION TPA is a photophysical process by which a molecule or material simultaneously absorbs two photons to reach an excited state. 5015

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Chart 1. Structures of 1−15

Notably, it has been well-established that the δ values of a molecule increase with the extent of intramolecular charge transfer (ICT),26−28,32,37,38 that is, the δ values of the electron donor−acceptor (D−A) dipoles, the D−π−D and D−A−D quadrupoles, and the two-dimensional octupoles can be increased by increasing the conjugation length and D−A strength (Figure 1c). Following this guideline, a variety of TP materials with large δ values (sometimes greater than 10 000 GM) have been developed.37 However, the enhanced ICT associated with these materials is usually accompanied by a reduction in the fluorescence, which is detrimental to the TP action cross section (Φδ), a measure of TP brightness. Therefore, it is essential to optimize both of these values to develop efficient TP fluorophores for use in biological TP probe applications.

which decreases as a function of 1/r2 (where r is the distance from the geometric focal point measured in the direction perpendicular to the laser beam), the efficiency of TPA is linearly proportional to 1/r4. This results in highly localized TP excitation at the focal point, which is an advantage in applications requiring high spatial resolution. The combined effects of this efficiency have led to a variety of applications, including microfabrication,26−31 three-dimensional optical data storage,27−33 optical power limiting,27,32,34 photodynamic therapy,28,32,35 and TPM imaging.13,18−21,36 A limitation of TPA is that it is much less efficient than OPA at low laser power levels. The ratio of OPA/TPA of a molecule can be expressed as n(1)/n(2) = 2[σ(ν)/δ(ν)](hν/I), where n(1) and n(2) are the number of molecules excited by one or two photons per unit time and unit volume, σ(ν) and δ(ν) are the OPA and TPA cross sections at frequency ν, I is the intensity of the excitation source, and hν is the photon energy.26 The ratio is equal to 1 at 800 nm for a molecule with σ(ν) = 104 M−1 cm−1, δ(ν) = 100 GM, and I = 10 GW/cm2, indicating that a maximum laser excitation greater than 10 GW/cm2 is needed for TPA to compete favorably with OPA. This is why the experimental verification of TPA was delayed until the advent of a high-energy pulsed laser. However, since such highpowered laser beams can damage the sample, organic materials with large δ values are needed to investigate the practical applications of TPA. Such molecules would allow TP excitation at a lower laser power, minimizing photodamage to the sample.

3. MODEL STUDIES ON TWO-PHOTON PROBES Along with the development of efficient TPA materials, parallel efforts have been focused on the application of TPA in bioimaging. Initial efforts were aimed at understanding whether well-established sensing mechanisms, such as ICT, photoinduced electron transfer (PeT), and resonance electron transfer (RET), operate in a TP mode.2 For example, the first TP probe (1) was designed on the basis of ICT and was developed by employing a D−A−D quadrupole as the TP reporter and aza-15-crown-5 as the metal ion receptor (Chart 1).39 It exhibited an emission maximum at 550 nm and strong 5016

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Table 1. Photophysical Data for 1−15 probe 1 1−Ca2+ 2 2−Mg2+ 3 3−Ag+ 4 4−Zn2+ 5 5−Zn2+ 6 6−Zn2+ 7 7−Zn2+ 8 8−Zn2+ 9 9−Cd2+ 10 10−Na+ 11 11−Zn2+ 12 12−K++Pb2+ 13 13−NaOH 14 14−TFA 15 15−Cys

solvent MeCN MeCN MeCN MeCN DMSO MeOH EtOH THF MeCN/CHCl3 MeCN DMF MeCN EtOH EtOH DMSO

a λ(1) max

λflmaxb

Φc

d λ(2) max

δe

Φδf

425 415 472 378 466 447 450 445 426 420 338 362 400 385 375 390 335 340 524 525 384 403 460 385 378, 397m 409, 430m 390 372, 391m 410 380

550 550 610 576 590 590 615 630 519 548 441 497 609 542 430 510 455 520 552 552 499 500 520 500 415, 439 550 505 408, 431 480 450

0.47 0.45 0.20 0.19 0.51 nai 0.47 nai 0.31 0.32 0.35 0.71 0.32 0.51 0.58 0.27 0.54 0.44 0.02 0.1 0.09 0.46 0.017 0.10 0.64 0.31 0.55 0.75 0.016 nai

760 880 810 810 810 810 780 780 750 720 690 730 820 820 nai 800 740 740 970 970 740 740 ndl 780 ≤705n 740, ≤ 705n ≤705n ≤705n 800 nai

120 25 2150 45 1120 340 30 nai 360 68 31 77 350 240 nai 193 95 520 86 216 530 470 ndl 998 ≥155 955, ≥1070 ≥490 ≥77 938 nai

56 11 430 9 571 nai 14 4 112 22 11 55 112 122 nai 52 51 229 1.7 21.6 48 216 ndl 100 ≥99 296, ≥ 332 ≥270 ≥58 15 nai

log Kg 4.3 (4.3) 3.8/2.4h 5.5/4.6 (5.8/4.4)j 6.0 (5.1) nai 5.6 8.0k 3.9 6.9/10.5j nai 9.6 (9.5)k nai 9.5o 3.5o nai

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. g Binding constants (K11), determined from 1:1 probe−metal complex. except where otherwise noted. The number in parentheses was measured by the two-photon process. hK11/K12, determined from 1:1/1:2 probe−metal complex. iNot available. jK11/K21, determined from 1:1/2:1 probe−metal complex. kK12, determined from 1:2 probe−metal complex. lNot determined. The two-photon excited fluorescence intensity was too weak for the cross section to be measured. mShoulder. nMaximum not yet reached. oThe pKa values. Data were measured in ethanol−water solution (3:1, v/v, 25 °C). a

two-photon excited fluorescence (TPEF), with a maximum TP action cross section (Φδmax) value of 56 GM at 760 nm in CH3CN (Table 1). Upon addition of Ca2+, the absorption spectrum was blue-shifted with a concomitant decrease in the absorption and emission intensities, presumably because of the reduced electron-donating ability of the nitrogen atom in the azacrown ether upon complexation. Similar spectral behavior was observed in TP mode.39 Moreover, the binding constant value (log K) of 1 for Ca2+, estimated from both OP and TP processes, was 4.3 (Table 1), indicating that a similar mechanism might be operating in both the OP and TP processes during the binding events. Almost simultaneously, another TP probe (2) derived from 2,5-dicyano-1,4-bis(styryl)benzene as the fluorophore and two aza-15-crown-5 as the receptor was reported in the literature.40 This investigation indicated that 2 has a red-shifted emission maximum with an enhanced Φδmax in CH3CN. In the presence of excess Mg2+, the TPEF of 2 decreased more sharply than that of 1 under comparable conditions (50- vs 5-fold) (Table 1), indicating that 2 is more sensitive to the changes in the electron-donating abilities of the azacrown ether. Further, 3 and 4 were developed using a similar strategy, except that 3,9-dithia6-azaundecane and bis(2-pyridyl)amine were employed as Ag+

and Zn2+ ion receptors, respectively (Chart 1).41,42 This strategy was further extended to 5, by incorporating a D−π− D quadrupole as the fluorophore and a tetraazacyclododecane as the Zn2+ ion receptor (Chart 1 and Table 1).43 Probes 6 and 7 were also derived by utilizing a D−π−A dipole as the fluorophores, but they contained tris(picolyl)amine and bis1,2,3-triazole as the Zn2+ ion receptors, respectively (Chart 1).44,45 Compound 6 displayed a gradual red shift in the absorption and emission maxima as well as an increase in the δmax value upon binding with Zn2+ in MeOH (Table 1), presumably due to enhanced ICT driven by the increased electron-withdrawing ability of the acceptor upon complexation. Similar bathochromic shifts were observed for tris[p-(4pyridylethyl)phenyl]amine (8) and bidentate phosphane oxide derivatives (9), which were developed to detect Zn2+ in THF and Cd2+ in MeCN/CHCl3, respectively.46,47 On the other hand, the absorption and emission maxima of 7 were blueshifted in the presence of Zn2+ ions in EtOH (Table 1), likely caused by the reduced ICT resulting from the decreased electron-donating ability. Consistent with this, the δmax value of 7 decreased by 1.5-fold upon complexation. The small increase in the Φδmax upon complexation has been attributed to the enhanced Φ value (Table 1). 5017

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Chart 2. Representative Fluorophores (16−24) Employed for the Design of Small-Molecule Two-Photon Probes

showed a blue-shifted and an enhanced Φδmax value as the consequent formation of thiazolidines reduced ICT and increased the Φ value. Taken together, these results indicate that the sensing mechanism typically utilized in the OP probes also functions in a TP mode, confirming that the basic principles developed for the design of OP probes can also be applied to the design of TP probes. It should be noted that all of the probes discussed in this section have only been tested in organic solvents due to their negligible water solubility. This hydrophobicity, therefore, prevented the application of these probes to living systems. However, while these model studies are limited, they have sufficiently outlined the basis of TP probe design, guiding the synthesis of probes specific for biological applications.

In addition to ICT-based probe design, the PeT mechanism was also used to develop TP “turn-on” probes, such as 10, which is used to detect Na+ ions.48 This probe was specifically developed by employing rhodol as the fluorophore and azacrown ether as the receptor, which are linked by a 1phenyl-1H-pyrazole group (Chart 1). This probe emitted weak fluorescence due to the partial PeT. The addition of Na+ to 10 in CH3CN increased the fluorescence as a result of blockage of the PeT (Table 1). A similar result was observed in TP mode, with an 11-fold increase in the TP brightness after the addition of Na+ (Table 1). Additional examples of TP turn-on probes include 11 and 12. The former is a TP turn-on probe for Zn2+ ions generated using a fluorene derivative as the fluorophore and 2-(2′hydroxyphenyl)benzoxazole (HPBO) as the Zn2+ ion receptor.49 This probe showed a 5-fold increase in the Φ value and a parallel increase in the Φδmax value upon complexation with Zn2+, presumably because of a reduction in vibrational relaxation upon binding. In contrast, 12 is a 1,3-alternate calix[4]arene derivative designed to detect Al3+ and Pb2+ based on the RET mechanism (Chart 1).50 As such, when excited at 780 nm, 12 emitted no TPEF in MeCN because the fluorescence was efficiently quenched by the adjacent fluorophores. Addition of Al3+ or Pb2+ caused an appreciable blue shift in the absorption spectrum and a significant increase in the TPEF intensity as a result of the attenuated RET. The TPEF intensity of 12−Pb2+ was further increased upon addition of K+. This outcome has been attributed to the allosteric effect induced by complexation of K+ with a crown loop, which further reduces the RET. Notably, as reaction-based TP probes, 13 and 14 were designed to detect pH using fluorene as the π-conjugating core and bis(4-vinylphenol) or bis(4-vinylaniline) moieties for the acid−base reaction sites, respectively (Chart 1).51 The deprotonation of the OH group of 13 appears to increase its electron-donating ability, causing the absorption and emission maxima of this probe to be red-shifted with a large increase in the δmax value under basic pH. On the other hand, the blueshifted spectra and the large decrease in the δmax value observed for 14 under acidic pH is a result of protonation at the amino group decreasing its electron-donating ability (Table 1). A similar result was observed in a model vesicle labeled with 13 at a different pH. Reaction-based probe 15 was generated using a triphenylamine derivative as the reporter and the aldehydes in the periphery as the reaction site for cysteine (Cys) and homocysteine (Hcy).52 This probe exhibited a Φδmax value of 15 GM at 800 nm in DMSO. In the presence of Cys or Hcy, 15

4. DESIGN OF TWO-PHOTON PROBES The model studies described in the previous section have established that TP probes can be designed using the same strategies employed for OP probes, except that fluorophores with significant Φδmax are needed. The requirements for a TP probe for biological targets can be summarized as follows: (i) a significant Φδmax value to obtain bright TPM images at low probe concentrations, (ii) appreciable water solubility to stain cells and tissues, (iii) a high specificity for the target analyte for selective detection, and (iv) high photostability for long-term imaging. To obtain a bright TPM image without causing appreciable photodamage to the sample at a laser power commonly used in the imaging experiment (1 GW/cm2 at the focal plane or ∼5 mW at the objective lens), the Φδmax of the probe should be greater than 50 GM. This can be achieved by increasing the donor−acceptor strength and intramolecular conjugation length, as stated above. The water solubility can be increased to a few micromolar, a concentration suitable to stain cells and tissues, by reducing molecular size and introducing water-solubilizing groups. The photostability can be increased by enclosing the conjugation bridge within the heterocycle. Representative fluorophores employed for the TP probes, the structures of which have been optimized to meet the abovementioned requirements, and their photophysical properties are summarized in Chart 2 and Table 2, respectively. Other fluorophores that have been employed to develop TP probes will be introduced in later sections. There are five types of TP probes in the literature. One is the TP tracer, developed by attaching either an organelle-targeting moiety or a protein or antibody to the fluorophore such that the targets can be detected by monitoring the TPEF (Figure 2). 5018

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The second type is comprised of TP probes for metal ions, protons, and small molecules constructed by linking the receptors for the analytes to the TP fluorophore either through a spacer or directly (Figure 2). Depending upon the sensing mechanism, they can be classified as turn-on, ratiometric, and reaction-based probes.2,7,53,54 The most common is the TP turn-on probe based on the PeT process, which undergoes a change in TPEF intensity upon binding with an analyte. The third type of TP probe that has been recently investigated is the TP turn-on probe with an internal reference. Probes of this type are typically designed by linking a TP turnon probe (FL, Ired) to the internal reference (IR, Iblue), which emits TPEF at a shorter wavelength, such that the efficiency of the Förster resonance energy transfer (FRET) from IR to FL is appreciably less than 100%, resulting in non-negligible Iblue. Notably, in this probe system, Ired will respond to the metal ion concentration and Iblue will remain constant, allowing the metal ion concentration to be quantitatively estimated by calculating the Ired/Iblue ratio. The fourth type is TP ratiometric probes, which respond with a red- or blue-shift of the TPEF spectra, depending on the respective change in the ICT during binding events, and are useful for quantitative detection of the analyte. Since the metal ion complexation and protonation reactions are fast and reversible processes, these probes can detect analytes in almost real time. The fifth type is the reaction-based TP probes for metal ions and small molecules, such as thiols, H2S, H2O2, and NO. The advantage of such probes is their high selectivity toward the

Table 2. Photophysical Data for 16−24 compd g

16 17h 18i 19j 20k 21l 22m 23n 24n

a λ(1) max

λflmaxb

Φc

d λ(2) max

δe

Φδf

413 364 360 362 408 368 382 434 452

556 496 470 497 535 555 570 514 538

0.29 0.76 0.98 0.24 0.20 0.12 0.56 0.34 0.50

820 780 780 750 810 740 770 860 870

290 122 153 170 260 633 100 814 877

84 93 150 170 52 76 56 276 438

λmax of the one-photon absorption spectra in nm. bλmax of the onephoton emission spectra in nm. cFluorescence quantum yield. dλmax of the two-photon excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gMeasured in micellar (10 mM SDS) solution (ref 101). h Estimated from probe 25 (ref 59). Data were measured in universal buffer (pH 7.0). iEstimated from probe 48 (ref 110). Data were measured in 30 mM MOPS (100 mM KCl, pH 7.2) in the presence of Ca2+ (2.5 mM). jEstimated from probe 129 (ref 257). Data were measured in PBS buffer (pH 7.4). The value of two-photon cross section measured in EtOH. kEstimated from probe 101 (ref 197). Data were measured in 100 mM PBS buffer (containing 5% CH3CN, pH 7.4) after addition of NO (100 μM). lEstimated from probe 41 (ref 100). Data were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM EGTA, pH 7.2) in the presence of Mg2+ (100 mM). m Estimated from probe 77 (ref 156). Data were measured in aqueous buffer at pH 10. nMeasured in PBS buffer (pH 11, 1% EtOH) (ref 172). a

Figure 2. Schematic diagram illustrating the sensing mechanism of (a) organelle-targeted, (b) turn-on, (c) turn-on with an internal reference, (d) ratiometric, and (e) reaction-based TP probes. 5019

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Chart 3. Structures of 25−33

Table 3. Photophysical Data for 25−33 probe 25 26 27 28 29 30 31 32 33

(AL1) (CLT-blue) (CLT-yellow) (BLT-blue) (LT1) (LT8) (9B-HVC) (SPHP) (FMT-green)

solvent

a λ(1) max

λflmaxb

Φc

d λ(2) max

δe

Φδf

bufferg buffer/EtOHh EtOH EtOH bufferi c-Hex MeCN bufferk EtOH

364 389 446 354 387 440 434 423 372

496 471 549 451 501 480 570 595 523

0.76 0.87 0.61 1.00 0.38 0.75 0.097 0.07 0.63

780 750 840 750 700j 790 810 890 750

122 57 77 160 1135j 2517 373 109 278

93 50 47 160 431.3j 1887 36 8 175

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. g Universal buffer (pH 7.0). hUniversal buffer/EtOH (1/1). iPBS buffer (pH 7.4). jTwo-photon values measured in toluene. kPBS buffer (pH 7.0). a

two organelles play critical roles in a myriad of cellular processes. To detect them in live cells or tissue by TPM, a variety of TP LysoTrackers (25−30) and MitoTrackers (31− 33) have been developed (Chart 3). The most common strategy used to develop TP LysoTrackers is to link an amino group, serving as the protonation site, to a fluorophore exhibiting significant TP action cross section, whereupon the protonated form will accumulate in the lysosomes after internalization. AL1 (25) is the first example of a TP LysoTracker, developed by linking the tertiary amine (pKa ≈ 10) to an acedan moiety through an amide bond.59 Compound 25 showed an emission maximum at 496 nm (Φ = 0.76) with the Φδmax value of 93 GM at 780 nm in universal buffer (pH 7.0), a value 9-fold larger than that of the commercial LysoTracker Red (LTR) (Table 3). The TPM image of macrophages colabeled with 25 and LTR revealed bright spots that overlapped well with the OPM image using

analyte. However, they cannot visualize dynamic concentration changes of the analyte because the reaction is irreversible. Moreover, the TPM image reflects the total concentration of analyte that has reacted with the probe until the point of TPEF detection. Therefore, reaction-based probes are useful for qualitative measurements only. Examples of TP probes developed for bioimaging applications are presented in later sections.

5. TWO-PHOTON PROBES FOR LYSOSOMES AND MITOCHONDRIA Lysosomes and mitochondria are essential organelles found in most eukaryotic cells. Lysosomes are acidic organelles (pH 4.5−5.5) that can serve to activate enzyme functions and protein degradation,55,56 while mitochondria supply the energy of the cells and are the primary site of oxygen consumption and the major source of reactive oxygen species (ROS).57,58 These 5020

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Figure 3. Images of a fresh rat hippocampal slice stained with 25. (a) Bright field image shows the CA1 and CA3 regions as well as the dentate gyrus (DG) upon 10× magnification. (b) 40 TPM images were accumulated along the z-direction at the depth of ∼100−250 μm. (c) 100× Magnification of CA3 regions at a depth of ∼120 μm. (d) Enlargement of the red box in part c shows the transport of acidic vesicles along the axon. Scale bars: 300 μm (b) and 30 μm (c). Modified with permission from ref 59. Copyright 2008 Wiley-VCH.

Figure 4. Images of HCT 116 cells incubated with 29: (a) DIC, (b) one-photon fluorescence image, (c) 3D reconstruction from overlaid TPM images, and (d) TPM image. (e, f) Dual-color TPM images of Raw 264.7 cells colabeled with 28 (green) and 33 (red). The cells were treated with rapamycin for (e) 0 h and (f) 2 h to induce autophagy. (g) Change in the TPEF intensities of the probe-labeled cells with time after treatment with rapamycin at 400−450 (28) and 550−600 nm (33). Modified with permission from (a−d) ref 62 (copyright 2010 American Chemical Society) and (e−g) ref 61 (copyright 2012 Wiley-VCH).

LTR. This result established the utility of 25 as a TP LysoTracker in live cells. The utility of 25 in live tissue imaging was also investigated. The TPM image of a fresh rat hippocampal slice labeled with 25 showed that acidic vesicles were more abundant in CA3 and dentate gyrus (DG) than in CA1 regions (Figure 3). Moreover, the real-time TPM images revealed that 25 could be used to monitor the transport of acidic vesicles at ∼120 μm depth for more than 1100 s in live tissue (Figure 3).

TP LysoTrackers emitting TPEF at different wavelength ranges (26−28) were developed by employing 2H-benzo[h]chromene-2-one (chromene) and 6-(benzo[d]oxazol-2′-yl)-2(N,N-dimethylamino)naphthalene (BODAN) as the TP fluorophores and a tertiary amine as the lysosomal targeting moiety.60,61 The photophysical properties of 26−28 were determined in model systems, which were chosen on the basis of similarities between the TPEF spectra as measured in the solvents and in live cells. Notably, 26 and 27 emitted TPEF of similar intensity at 471 and 549 nm, respectively, while 28 5021

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emitted fluorescence that was 3-fold stronger than that of these other TP LysoTrackers at 451 nm (Table 3). The utility of 26− 28 in cell and tissue imaging was demonstrated.60,61 LT1 (29) is a fluorene derivative possessing two 4(dimethylamino)phenylacetylene groups at the 2,7-positions as the lysosomal targeting moiety and bis(oligoethylene glycol) groups at the 9-position as the water-solubilizing group.62 In PBS buffer, an emission maximum was observed at 501 nm (Φ = 0.38) for this probe, while a larger Φδmax value was observed in toluene (Table 3). The lysosomal specificity of 29 was demonstrated by colocalization experiments.62 Probe 29 showed low cytotoxicity and high photostability in live cells and was successfully utilized in 3D TPM imaging of individual lysosomes in HCT 116 cells (Figure 4a−d). LT8 (30), having the largest MW among the fluorene derivatives, emitted fluorescence at 480 nm (Φ = 0.75), with the Φδmax value of 1887 GM at 790 nm in cyclohexane (Table 3).63,64 To load 30 into cells, an encapsulation method using Pluronic F 108NF micelles, a block copolymer based on ethylene oxide and propylene oxide, was employed. The TPM image of HCT 116 cells incubated with 30-encapsulated micelles showed that 30 was taken up by cells with high lysosomal selectivity. The design strategy for the TP MitoTrackers is almost the same as that for TP LysoTrackers except that pyridinium and/ or triphenylphosphonium (TPP) ions are used as the mitochondrial targeting moiety.65,66 Since mitochondria have a negative membrane potential, cationic species are expected to be localized within mitochondria. 31 is a carbazole derivative having a pyridinium ion as the fluorophore and mitochondriatargeting moiety.67 Probe 31 emitted fluorescence at 570 nm (Φ = 0.097) with the Φδmax value of 36 GM at 810 nm in MeCN (Table 3). It showed mitochondrial specificity and low cytotoxicity in live SiHa cells. More recently, a TP MitoTracker containing both pyridinium and TPP ions (32) was reported.68 Probe 32 emitted fluorescence at 595 nm (Φ = 0.07) with the Φδmax value of 8 GM at 890 nm in PBS buffer (Table 3). It showed mitochondria-targeting ability and low cytotoxicity in live HK-1 cells. To simultaneously visualize both lysosomal and mitochondrial activities by dual-color TPM imaging, TP trackers that emit TPEF at widely separated wavelengths are required. To meet such a demand, a fluorene derivative containing TPP (33) was developed.61 Compound 33 emitted TPEF at 540 nm in RAW 264.7 cells, which was separated from the emission fluorescence of 28 (456 nm) by more than 80 nm, and it showed mitochondrial specificity, low cytotoxicity, high photostability, and a Φδmax value of 175 GM at 750 nm in EtOH (Table 3). Hence, it was possible to study autophagy, a tightly regulated catabolic process that degrades old and damaged mitochondria via the lysosomes,69,70 by dual-color TPM imaging using detection windows at 400−450 nm (28) and 550−600 nm (33). The TPM image of RAW 264.7 cells colabeled with 28 and 33 revealed the lysosomes (green spots) and mitochondria (red spots), which did not overlap when the images were merged (Figure 4e). Upon treatment with rapamycin, a reagent that induces autophagy, the intensity of the green spots increased gradually with concomitant decreases in red spot intensity (Figure 4e−g). This outcome indicates the activation of lysosomes and breakdown of mitochondria, a result consistent with the progression of autophagy. Hence, the utility of 28 and 33 in dual-color TPM imaging in live cells was clearly demonstrated. The utility of 28 and 33 in dual-color imaging of tissue was also established.61

6. TWO-PHOTON PROBES FOR PLASMA MEMBRANES There is ongoing controversy regarding the existence of rigid domains (termed lipid rafts) in the plasma membrane.71 The proponents of the lipid raft model posited that these exist within the cell membrane and are involved in various cellular functions, including signal transduction, pathogen invasion, cholesterol homeostasis, and neurodegenerative diseases.72−75 However, many scientists do not believe in such domains and/ or their biological relevance. To visualize membrane heterogeneity, generalized polarization (GP) imaging using 6dodecanoyl-2-(dimethylamino)naphthalene (laurdan) has most frequently been used.76 Laurdan, found to be an excellent polarity probe for the model membrane composed of 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),77 has been used for more than 2 decades. Unfortunately, it did not work as well in cell imaging, probably because of its negligible water solubility and/or membrane-staining ability. In fact, some of the results from cell imaging using laurdan were difficult to reproduce.78 Overcoming such problems required the development of a new TP probe with enhanced membrane-staining ability and higher sensitivity to membrane polarity. To this end, 6dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene [CL (34), Chart 4], having a carboxylic acid Chart 4. Structures of 34−38

headgroup, was developed.78 Compound 34 showed enhanced water solubility, higher sensitivity to the solvent and to vesicle polarity, and larger Φδmax value than laurdan (Table 4). Moreover, the GP curve of 34-labeled A431 cells was symmetrical and could be deconvoluted into two curves of similar shape (the GP values range from −1.0 to 1.0; larger numbers indicating a more hydrophobic phase) (Figure 5a,b).78,79 When the cells were treated with methyl-βcyclodextrin (MβCD), a lipid-raft-destroying reagent, the high GP curve decreased dramatically with negligible change in the low GP curve (Figure 5c,d). Further, the colocalization experiments with cells colabeled with 34 and BODIPY-GM1, a well-known one-photon fluorescent marker for lipid rafts, confirmed that the high GP domains were indeed lipid rafts. These results established that 34 can reflect the cellular 5022

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near the lipid head groups.78 The effect of hydrocarbon chain length on probe properties was also studied. CH (35), having a 6-carbon chain in the acyl moiety, did not stain the membrane, probably because of its high water solubility; CS (36), having an 18-carbon chain, exhibited a greater tendency to be located in the plasma membrane; and 34, having a 12-carbon chain, showed the optimum staining ability.79 The utility of 34 in cell biology was demonstrated by other researchers.80−83 To directly visualize lipid rafts without invoking GP values, it was necessary to develop a TP probe that emitted TPEF only in the rigid domain. For this purpose, TP turn-on probes for lipid rafts [CL2 (37) and SL2 (38)] possessing longer conjugation lengths than that of 34 were developed (Chart 4).84,85 Compound 37 showed large bathochromic and hypochromic shifts with the solvent polarity. Also, the Φδmax value was larger in a more hydrophobic environment, larger in DMF than in EtOH by 6-fold, and larger in DPPC than in 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) by 8-fold (Table 4). This outcome predicted that 37 would emit TPEF only in the lipid rafts in the cell membrane. Indeed, the TPM image of macrophages labeled with 37 showed a bright domain, which disappeared upon treatment with MβCD and returned to near basal level upon addition of cholesterol.84 This outcome confirmed that 37 is a TP turn-on probe for lipid rafts. However, 37 had one shortcoming: it internalized into the cytoplasm upon prolonged incubation. To overcome this problem, a new probe having a sulfonate headgroup (38) was developed (Chart 4).84,85 The photophysical properties of 38 were nearly identical to those of 37 (Table 4).85 However, it emitted TPEF only in the plasma membrane, showed minimum internalization, and allowed direct visualization of the lipid rafts (Figure 5e). The utility of 38 in live tissue imaging was demonstrated (Figure 5f).

Table 4. Photophysical Data for 34−38 probe

solvent

a λ(1) max

λflmaxb

Φc

d λ(2) max

δe

Φδf

34 (CL)

DMF EtOH DMF EtOH DMF EtOH DMF EtOH DMF EtOH

369 385 370 383 372 383 392 401 401 402

444 493 444 493 444 492 535 580 545 576

0.36 0.43 0.18 0.33 1.00 0.96 0.51 0.09 0.70 0.12

780 780 780 780 780 780 800 800 800 800

170 150 190 150 90 85 230 250 250 330

60g 65g 35 50 90 80 120 20 175 40

35 (CH) 36 (CS) 37 (CL2) 38 (SL2)

λmax of the one-photon absorption spectra in nm. bλmax of the onephoton emission spectra in nm. cFluorescence quantum yield. dλmax of the two-photon excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gΦδ values of laurdan was 50 and 60 GM in DMF and EtOH, respectively. a

7. TWO-PHOTON PROBES FOR METAL IONS Cellular metal ions play important roles in many physiological and pathological processes. For proper cell function, metal ion homeostasis must be tightly controlled. Disruption of this balance can cause aging and disorders including cancer, neurodegenerative diseases, and diabetes.86−88 Metal ions are also involved in pollution. Heavy metal ions can enter the environment through natural causes as well as human activities and can accumulate in animals and fish.89−91 To understand the roles of these ions in biology and the environment, it is important to develop TP probes that can detect metal ions at the live cell, tissue, and organism levels. In this section, we will summarize the TP probes for biologically relevant metal ions such as Na+, Mg2+, Ca2+, and Zn2+, and other heavy metal ions related to pollution. 7.1. Two-Photon Probes for Sodium Ions

Figure 5. Reconstructed GP images from TPM images of A431 cells labeled with 34 (a) before and (c) after treatment with MβCD and (b, d) the corresponding GP distribution curves. Pseudocolored TPM images of (e) 293T cells labeled with 38 and (f) the dentate gyrus (DG) layer of a fresh rat hippocampal slice. The TPM images were collected upon excitation at (a, c) 780 nm and (e, f) 800 nm with femtosecond pulses. Scale bars: (a, c) 10 μm, (e) 30 μm, and (f) 75 μm. Modified with permission from refs 78 and 85. Copyright 2007 and 2011, respectively, Wiley-VCH.

Sodium ions modulate many physiological processes in the mammalian body, including electrolyte levels, cation transport, and cell volume.92−94 The concentrations of intra- and extracellular free Na+ ions are in the range of 5−30 mM and more than 100 mM, respectively. The intracellular free Na+ concentration ([Na+]i) is maintained by the activities of various ion-transport systems, such as the Na+/Ca2+ exchanger (NCX), the Na+/H+ exchanger, Na+ channels, and Na+/K+-ATPase.92−94 To detect the [Na+]i, SBFI and Sodium Green (SG) have been utilized most frequently.7 However, they suffer from short excitation wavelenths and poor cell loading. To overcome these problems, small-molecule TP probes (39 and

environment more accurately than laurdan, presumably because it can be aligned in parallel to lipid molecules, with its hydrocarbon tails in the lipid bilayers and its carboxylate moiety 5023

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Chart 5. Structures of 39−50

40) derived from acedan as the fluorophore and 1,7-diaza-15crown-5 as the Na+ ion receptor were developed (Chart 5). ANa1 (39) is a TP turn-on probe with a TP fluorescence enhancement factor (TPFEF) value of 8 and high selectivity for Na+ over K+, as indicated by its disassociation constant (Kd) values (20 and 280 mM for Na+ and K+, respectively).95 Further, the Φδmax value observed for the 39−Na+ complex was larger than those measured for both SG and SBFI by 3−5-fold under the same conditions (Table 5). The TPM image of 39labeled HeLa cells was much brighter than those of SG−AMor SBFI−AM-labeled cells, presumably because of the combined effects of a larger Φδmax and increased cell permeability. Compound 40 is a more sensitive TP probe with a 2-fold larger TPFEF value than that of 39.96 The enhanced TPFEF was attributed to the prolinamide linker, which may have facilitated the PeT from the receptor to the excited state fluorophore by forcing the two moieties into a more restricted conformation. The utility of 40 in live cell and tissue imaging has been demonstrated.96

Mg2, presumably as a result of blockage of the PeT process upon complexation with Mg2+. The TPFEF was 17 in the presence of excess Mg2+ in a Tris buffer solution (10 mM, pH 7.05). The Kd values of 41 for Mg2+ and Ca2+ ions were 1.4 mM and 9.0 μM, respectively, which were nearly identical to those measured for the TP processes (Table 5). Since the [Mg2+]i (0.1−6.0 mM) is much higher than [Ca2+]i (0.01−1 μM), 41 can detect the [Mg2+]i with minimum interference by Ca2+ and other metal ions. Notably, the Φδmax value of the 41−Mg2+ complex was 7-fold larger than those observed for two wellknown Mg2+ indicator dyes, Magnesium Green (MgG) and Mag-fura-2, under the same conditions (Table 5). This outcome predicts that the TPM image of cells labeled with 41−AM should be much brighter than those labeled with the commercial probes. Indeed, the TPM images of 41−AMlabeled Hep3B cells were bright.100 Moreover, the TPM image of 41−AM-labeled fresh mouse hippocampal slices resolved the [Mg2+]i distribution in the pyramidal neuron layer of the CA1 region (Figure 6), thereby demonstrating the utility of 41 in live tissue imaging. CMg1 (42) was then developed by integrating β-keto acid as the Mg2+ receptor in the D−A substituted chromene, while a small MW was maintained for optimal cell permeability.101 In an aqueous micellar solution ([SDS] = 10 mM), 42 showed an emission maximum at 556 nm and a 3-fold turn-on response upon binding with Mg2+. The Kd values of 42 for Mg2+ and Ca2+ were 1.3 and 3.6 mM, respectively (Table 5), indicating much higher selectivity for Mg2+ over Ca2+ than that of 41. Similar to other TP turn-on probes, this probe had a 20-fold larger Φδmax value than those measured for MgG and Mag-fura2 in the presence of excess Mg2+. The utility of 42 in live cell and tissue imaging has also been demonstrated.101

7.2. Two-Photon Probes for Magnesium Ions

Magnesium ions are one of the most abundant divalent cations in cells and are involved in many cellular processes, such as cell proliferation, enzymatic reactions, and signal transduction.97−99 To visualize intracellular free Mg2+ ([Mg2+]i) by TPM, various TP probes for Mg2+ (41−43) have been developed (Chart 5). AMg1 (41) is the first example of a TP turn-on probe for metal ions.100 Compound 41 was designed by a simple and expandable strategy; o-aminophenol-N,N,O-triacetic acid (APTRA), a well-known Mg2+ receptor, was linked to acedan, a well-known TP fluorophore, through an amide bond (Chart 5). To enhance its cell permeability, the carboxylate groups were converted into the acetoxymethyl (AM) ester (41−AM). The fluorescence intensity of 41 increased in the presence of 5024

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Table 5. Photophysical Data for 39−50 probe 39, ANa1 39−Na+ 40, ANa2 40−Na+ 41, AMg1 41−Mg2+ 42, CMg1 42−Mg2+ 43, FMg2 43−Mg2+ 44, ACa1 44−Ca2+ 45, ACa2 45−Ca2+ 46, ACaL 46−Ca2+ 47, ACaLN 47−Ca2+ 48, BCaM 48−Ca2+ 49, TP-BAPTA 50, TP-CN-BAPTA

solvent h

H2O

H2Oj H2Ok H2Ol H2Om H2On H2On H2On H2On H2Oo DMF DMF

a λ(1) max

367 367 366 366 365 365 413 443 368 368 365 365 362 362 369 372 364 354 360 360 370 434

λflmaxb

Φc

500 500 500 500 498 498 556 559 555 555 498 498 495 495 500 502 498 497 470 470 440 564

0.08 0.65 0.023 0.35 0.040 0.58 0.29 0.28 0.0058 0.12 0.012 0.49 0.010 0.42 0.0037 0.043 0.0013 0.018 0.070 0.98 0.35 0.12

d λ(2) max i

nd 780 750 750 ndi 780 820 880 ndi 740 ndi 780 ndi 780 ndi 780 ndi 750 ndi 780 800 800

δe i

nd 146 130 237 ndi 215 290 382 ndi 633 ndi 230 ndi 210 ndi ndi ndi 153 36 917

Φδf i

nd 95 3 83 ndi 125 84 107 ndi 76 ndi 110 ndi 90 ndi 90 ndi 20 ndi 150 10 110

Kdg 20 mM (20 mM) 21.6 mM (22 mM) 1.4 mM (1.6 mM) 1.3 mM (1.7 mM) 1.4 mM (1.7 mM) 0.27 μM (0.25 μM) 0.14 μM (0.16 μM) 45 nM (41 nM) 2.1 μM (1.9 μM) 90 μM (89 μM) 51 nM 39 nM

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gKd values measured by one-photon mode. The values in parentheses are measured by two-photon mode. hData were measured in 10 mM MOPS buffer ([Na+] + [K+] = 135 mM, pH 7.0) in the absence and presence (135 mM) of Na+. iNot determined. The two-photon excited fluorescence intensity was too weak for the cross section to be measured accurately. jData were measured in 10 mM MOPS buffer ([Na+] + [K+] = 135 mM, pH 7.0) in the absence and presence (500 mM) of Na+. kData were measured in 10 mM Tris buffer (100 mM KCl, 20 mM NaCl, 1 mM EGTA, pH 7.05) in the absence and presence (50 mM) of Mg2+. lData were measured in micellar solution (10 mM SDS) in the absence and presence (35 mM) of Mg2+. m Data were measured in 30 mM MOPS (100 mM KCl, 10 mM EGTA, pH 7.2) in the absence and presence (100 mM) of Mg2+. nData were measured in 30 mM MOPS (100 mM KCl, 10 mM EGTA, pH 7.2) in the absence and presence (39 μM) of Ca2+. oData were measured in 30 mM MOPS (100 mM KCl, pH 7.2) in the absence and presence (2.5 mM) of Ca2+. a

utility of 43 in dual-color imaging of Mg2+/Ca2+ activities in live cells and tissues has been demonstrated (see below).102 7.3. Two-Photon Probes for Calcium Ions

Calcium ions are ubiquitous and versatile intracellular messengers and are essential regulators of many cellular processes.103−105 In animal cells, calcium ion levels are tightly controlled by the coordinated actions of various calcium ion channels and transporters. The cytosolic Ca2+ concentration ([Ca2+]c) ranges from about 100 nM at rest to 1 μM upon activation, and the near-membrane Ca2+ concentration ([Ca2+]mem) can reach values larger than 100 μM upon activation.105 To understand the roles of Ca2+ in biology, various TP probes derived from different fluorophores along with O,O′-bis(2-aminophenyl)ethylene glycol-N,N,N′,N′-tetraacetic acid (BAPTA) as the Ca2+ receptor have been developed (44−50, Chart 5). Compounds 44 and 45 are TP turn-on probes for cytosolic Ca2+ developed by linking BAPTA to acedan through a glycinamide spacer.106,107 They showed Kd values of 0.27 and 0.14 μM with fluorescence enhancement factor (FEF) values of 41 and 40, respectively (Table 5). They were pH-independent in the physiologically relevant pH range and showed high selectivity for Ca2+ over other competing metal ions with the exception of Zn2+, which is a limitation of BAPTA. The Φδmax values measured for these probe−Ca2+ complexes were also over 2.5-fold higher than those found for both Calcium Green

Figure 6. (a) TPM images of a fresh mouse hippocampal slice stained with 41−AM at a depth of ∼270 μm. Images taken with magnification at 10×. (b) Magnification at 100× shows CA1 pyramidal neurons at a depth of ∼150 μm. Scale bars: 300 μm (a) and 30 μm (b). The TPM images were collected at 500−620 nm upon excitation at 780 nm with femtosecond pulses. Modified with permission from ref 100. Copyright 2007 Wiley-VCH.

To simultaneously detect two targets by TPM, it is crucial to develop TP probes that emit TPEF at widely different wavelength ranges. To meet such a demand, a TP probe derived from fluorene and APTRA [FMg2 (43)] was developed (Chart 5).102 Compound 43 showed an emission maximum at 555 nm in MOPS buffer (30 mM, pH 7.2), which was ca. 60 nm red-shifted from that of 41. It showed a nearly identical Kd value to that of 41 as well as a TPFEF value of 24, a significantly enhanced Φδmax value in the presence of excess Mg2+, low cytotoxicity, and high photostability (Table 5). The 5025

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and Fura-2, two Ca2+ indicator dyes used in biological applications. The TPM images of 44−AM-labeled astrocytes were bright, as expected from the significant Φδmax value.106 Moreover, the spontaneous Ca2+ waves could be visualized for more than 1000 s in 44−AM-labeled hypothalamic slices (Figure 7) and for more than 4000 s in 45−AM-labeled tissue,107 without appreciable decay. These results confirmed the utility of these probes in TPM imaging.

localization to the plasma membrane due to its favorable hydrophobic interactions with the lipid molecules in the membrane. In fact, TPM imaging of live cells labeled with 48 clearly revealed the Ca 2+ distribution in the plasma membrane.110 Furthermore, 48 showed high Ca2+ selectivity along with a TPFEF value of 14 and a large Φδmax value in the presence of excess Ca2+ (Table 5). The Kd values of 48 for Ca2+ measured in digitonin-treated HeLa cells was 78 μM, which was within the range of [Ca2+]mem in the cells. Further, 48 showed an emission maximum at 450 nm, which was well-separated from that of 43 (525 nm). These characteristics allowed simultaneous detection of [Ca2+]mem and [Mg2+]i activities in the cells and tissues colabeled with 48 and 43 by dual-color TPM imaging.97 The TPM image of the HepG2 cells colabeled with 48 and 43−AM displayed the distribution of [Ca2+]mem and [Mg2+]i (Figure 8a−c). The TPEF intensity in the membrane increased sharply upon treatment with epidermal growth factor (EGF) and calcimycin and then decreased to the baseline level. A similar result was observed in the cytoplasm, albeit at a slower rate. This outcome indicates that the EGF-induced influx of Ca2+ occurred at a faster rate than that of Mg2+. The distributions of [Ca2+]mem and [Mg2+]i deep inside a fresh rat hippocampal slice could also be visualized by dual-color TPM imaging (Figure 8d,e).110 Compounds 49 and 50 were developed by a different strategy: introduction of BAPTA directly into the fluorophore (Chart 5).111 These TP probes appeared to utilize a turn-off behavior in response to Ca2+, in association with larger Kd values (Table 5). Further, the Φδmax value of the 49−Ca2+ complex was very small, while that of 50−Ca2+ was 110 GM at 800 nm in DMF. The utility of 50 in cell imaging has been demonstrated.111

Figure 7. TPM images of a fresh rat hypothalamic slice stained with 44−AM taken after (a) 195 and (b) 214 s. Magnification at 100× shows the hypothalamic area at a depth of ∼170 μM. (c) Spontaneous Ca2+ transients recorded in the soma (1), astrocyte process (2), and a neighboring cell (3). The TPM images were collected at 500−620 nm upon excitation at 780 nm with femtosecond pulses. Scale bar: 30 μm. Modified with permission from ref 106. Copyright 2007 Wiley-VCH.

7.4. Two-Photon Probes for Copper Ions

Copper is required in the body as a cofactor for many metalloenzymes, including superoxide dismutase, cytochrome c oxidase, and tyrosinase.112 Further, the disruption of copper ion homeostasis can result in the development of Menkes, Wilson, Alzheimer’s, and prion diseases, while a high levels have also been shown to be toxic, causing gastrointestinal disorders and liver or kidney damage.113 Thus, two TP Cu2+ ion detection probes have been developed, characterized, and reported in the literature (51 and 52, Chart 6) and have been used in living systems in order to better understand the biological roles of this specific metal. Compound 51 is a TP probe for Cu2+ with an internal reference.114 The internal Cu2+ reference component that is exploited in this probe is a chromene derivative (IR, blue emission, Iblue) that is linked via a piperazine spacer to a coumarin derivative having a 2-picolylmethylamide moiety that is used to recognize Cu2+ (FL, red emission, Ired) (Chart 6). Notably, during probe validation, 51 was shown to be highly selective for Cu2+ ions, while also functioning independently of pH and producing a significantly bright emission spectra following TP excitation (Table 6). Upon addition of Cu2+ to 51 in an EtOH/H2O (9:1) solution, Ired decreased by 10-fold without an appreciable change being observed for Iblue. The titration curve indicates a Kd value of 21 μM, while the plot of the Ired/Iblue ratio vs [Cu2+] was linear in the range from 0 to 25 μM, indicating that accurate measurements of [Cu2+] are possible in this concentration range. For example, in HeLa cells, the average ratio (Ired/Iblue) calculated using TPM imaging of

To detect [Ca2+]mem, 46 and 47 were developed using the same strategy as above, except that a long chain hydrocarbon tail was introduced to enhance the hydrophobic interactions with the lipid molecules in the plasma membrane.108,109 Compounds 46 and 47 showed Kd values of 0.27 and 0.14 μM with FEF values of 12 and 14, respectively (Table 5). They functioned independently of pH, at least within a physiologically relevant range, and showed high Ca2+ selectivity and a significant Φδmax value in the presence of excess Ca2+. The TPM images of 47-labeled HT22 cells displayed bright TPEF in the plasma membrane that could be attributed to the [Ca2+]mem.109 The TPEF intensity increased slowly upon addition of histamine, a reagent that stimulates the cells to release [Ca2+]c from intracellular Ca2+ stores, and decreased to the basal level within 300 s. A similar result was observed upon addition of CaCl2, except that the response was faster and larger. The utility of 47 in tissue imaging was demonstrated.109 BCaM (48) is another TP probe for [Ca2+]mem developed with BODAN as the fluorophore and 2-(2′-morpholino-2′oxoethoxy)-N,N-bis(hydroxycarbonylmethyl)aniline (MOBHA) as the Ca2+ receptor (Chart 5).110 It was expected that the cholesterol-like structure of BODAN would lead to its 5026

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Figure 8. (a, b) Dual-channel TPM images of HepG2 cells colabeled with 48 and 43−AM collected at 400−450 [48, channel 1 (Ch1)] and 525− 600 nm [43−AM, channel 2 (Ch2)], respectively. TPM images were obtained in PBS buffer (a), 200 s after stimulation with calcimycin and EGF in the presence of Mg2+ (b). (c) Time course of TPEF at designated positions A (green curve) and B (red curve) in part b after stimulation. (d) Ten TPM images collected at Ch1 and Ch2 along the z-direction at depths of approximately 100−200 μm were accumulated and then merged. (e) TPM images of CA1 regions collected at Ch1 and Ch2 at a depth of about 100 μm at 100× magnification and then merged. Scale bars: 15 μm (a), 300 μm (d), and 30 μm (e). Modified with permission from ref 102. Copyright 2012 American Chemical Society.

respectively. Similar results were observed when 51 was used to label rat brain tissues, further highlighting use of 51 in detecting [Cu2+] with statistical significance in fresh whole tissues. Notably, ratiometric TPM imaging using this probe has also been investigated as a biomedically effective tool for estimating [Cu2+] in human colon tissues (see section 15, “Two-Photon Probes for Medical Applications”). On the other hand, 52, a ratiometric TP probe for Cu2+ detection based on through-bond energy transfer (TBET), was designed by directly conjugating BODAN (as the TBET donor, green emission) and rhodamine spirolactam (as the TBET acceptor, red emission) (Chart 6).115 When tested in an EtOH/H2O (1:9) solution, 52 emitted green fluorescence from the BODAN moiety, which, upon the addition of Cu2+, gradually disappeared with a concomitant increase in the redshifted emission. It is thought that this increase in emission from the rhodamine moiety occurs as a result of Cu2+-induced ring-opening. Similar to 51, 52 showed a high selectivity for Cu2+, pH independency, and significant TP brightness (Table 6). Moreover, the Cu2+ distribution in 52-labeled HeLa cells as well as labeled slices of frozen rat liver pretreated with Cu2+ could be visualized using TPM in the 450−530 and 540−650 nm detection windows (Figure 9).

Chart 6. Structures of 51 and 52

51-labeled cells was 9.9, corresponding to a [Cu2+] value of 0.0 μM. Notably, the ratio decreased to 6.5 upon the addition of pyrrolidine dithiocarbamate (PDTC), a mediator that aids Cu2+ accumulation inside the cell, but was increased to 9.8 upon treatment with EDTA, which effectively removes Cu2+ ions. These ratios corresponded to [Cu2+] values of 15 and 0.0 μM, Table 6. Photophysical Data for 51 and 52 probe 51, ACCu2 51−Cu2+ 52, Np−Rh 52−Cu2+

a λ(1) max

solvent i

EtOH/H2O

EtOH/H2Oj

379/465 nah 395 525

λflmaxb

Φc

585 585 475 575

0.22 nah nah nah

d λ(2) max

750 nah 780 nah

δe h

na nah 115 nah

Φδf

Kdg

32 nah nah nah

21 μM (22 μM) nah

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gKd values measured by one-photon mode. The values in parentheses are measured by two-photon mode. hNot available. iData were measured in EtOH/ HEPES (9/1 v/v, pH 7.0) in the absence and presence (200 μM) of Cu2+. jData were measured in EtOH/Tris-HCl (1/9 v/v, pH 7.4) in the absence and presence (250 μM) of Cu2+. a

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Zn2+, and decreased upon addition of N,N,N′,N′-tetrakis(2pyridyl)ethylenediamine (TPEN), a membrane-permeable Zn2+ chelator that can effectively remove [Zn2+]i.126 These results indicated that the bright regions in the TPM images reflected the [Zn2+]i distribution. The utility of other probes in cell imaging was established. The TPM images of a fresh rat hippocampal slice labeled with 54 are shown in Figure 10. The image revealed the stratum lucidum (SL) of CA3 and the hilus (H) of the dentate gyrus (DG) (Figure 10a−c). The TPEF intensity increased upon addition of KCl, a membrane depolarizer that causes the release of Zn2+ (Figure 10c,d), and decreased after treatment with TPEN. Moreover, the image of the SL taken at higher magnification revealed that [Zn2+]i is accumulated in the mossy fiber axon terminals of the pyramidal neurons (Figure 10b). These results confirmed the utility of 54 in tissue imaging. The utility of other probes in tissue imaging was also demonstrated.123 57 is a TP turn-on probe for Zn2+ with an exceptionally large Kd value. It was developed by using dicyanostilbene as the fluorophore and 4-(pyridine-2-ylmethyl)piperazine as the Zn2+ receptor.127 57 showed an emission maximum at 610 nm in MOPS buffer with a Φδmax value of 8 GM, which increased to 580 GM upon complexation with Zn2+. The Kd value of 57 for Zn2+ was 0.52 μM (Table 7), which was larger by ∼1000-fold than those of DPEN derivatives. The TPEF intensity of 57labeled mouse fibroblast cells increased upon addition of SNOC and decreased upon treatment with TPEN. The TPM imaging of a 57-labeled mouse brain tissue slice was also reported.127 To date, three ratiometric TP probes for Zn2+ (58−60) have been reported. Compound 58 is an acedan-based TP probe having N,N-di(2-picolyl)amine (DPA) at the acyl moiety (Chart 7).128 The emission spectrum was red-shifted by 24 nm upon binding with Zn2+ , presumably because of coordination of the acyl oxygen by Zn2+, which would have enhanced the acceptor ability and the ICT. The Kd value measured for this probe was 20 nM, a value 20-fold larger than that of 53, and was associated with Φδmax values that were 20− 26-fold smaller than that observed for 53 in the presence of excess Zn2+. The larger Kd value can be attributed to the reduced basicity of the tertiary amino group in the receptor by the CO group at the β-position, while the smaller Φδmax values are due to the smaller Φ values as a result of the enhanced ICT (see above). The utility of this probe in cell imaging has been demonstrated.128 Compound 59 was derived from 6-[(p-methoxyphenyl)ethynyl]quinoline as the fluorophore and DPA as the receptor (Chart 7).129 In MeOH/H2O (1:1), the fluorescence spectrum was red-shifted from 412 to 493 nm upon complexation with Zn2+, presumably because of the enhanced ICT from coordination of the Zn2+ with the quinoline nitrogen. The Kd value was 0.45 nM. In the presence of excess Zn2+, the I493/I412 ratio increased by 14-fold, and the Φδmax value increased from 7 GM at 710 nm to 125 GM at 720 nm (Table 7). Ratiometric TPM images of 59-labeled HeLa cells have been reported.129 In contrast, probe 60 was developed by employing a D−A− D quadrupole possessing a bispyridine core as the Zn2+ binding site, two carbazole moieties as electron donors, and oxyethylene side chains to enhance probe solubility (Chart 7).130 In a 1:1 MeCN/H2O solution, 60 showed a red-shifted emission spectra (from 530 to 610 nm) upon binding to Zn2+ along with a 9-fold increase in the TP brightness (Table 7).

Figure 9. Two-photon images of a slice of frozen rat liver incubated with 52 (Np−Rh) for 60 min (a, b), followed by incubation with 100 μM Cu2+ for another 60 min (c, d). The images were collected at 450−530 nm (green channel; a, c) and 540−650 nm (red channel; b, d) upon excitation with femtosecond pulses of 780 nm light. Scale bars: 100 μm. Modified with permission from ref 115. Copyright 2014 American Chemical Society.

7.5. Two-Photon Probes for Zinc Ions

Zinc ions are an active component in enzymes, proteins, and gene transcription.116,117 The total Zn2+ ion concentration in a mammalian cell is approximately 0.2 mM, of which a trace amount exists as intracellular free Zn2+ ([Zn2+]i).118 In the brain, Zn2+ ions are mainly accumulated in synaptic vesicles and play important roles in neurotransmission.119 The [Zn2+]i homeostasis is tightly controlled by the activities of Zn transporters that control import/export processes and of subcellular organelles such as mitochondria and the endoplasmic reticulum. An imbalance in [Zn2+]i homeostasis can cause neurological disorders, such as Alzheimer’s and Parkinson’s diseases.120,121 To understand the physiological roles of the Zn2+, it is a crucial task to monitor the [Zn2+]i distribution at the subcellular level. For this purpose, a variety of TP probes for Zn2+ ions have been developed (53−64, Chart 7). Compounds 53−56 are TP turn-on probes for [Zn2+]i, derived using acedan as the fluorophore122,123 and N,N-di(2picolyl)ethylenediamine (DPEN) derivatives as the Zn2+ chelator,124,125 and they showed Kd values in the range of 0.5−21 nM (Table 7). The Kd value decreased and the TPFEF value increased by inclusion of a 2-OMe group in the phenylenediamine moiety (53 vs 54). On the other hand, the Kd value was increased by a 2-methyl group in the pyridyl moiety (54 vs 55) and a longer spacer in the receptor (54 vs 56). Compounds 53−56 showed high selectivity for Zn2+ and were pH-independent in the physiologically relevant pH range. The Φδmax values of probe−Zn2+ complexes of 53−56 were 85−110 GM (Table 7), which were larger by 4−24-fold than those of TSQ and FluZin-3.122,123 The TPM images of 54-labeled 293 cells were bright. The TPEF intensity increased after treatment with S-nitrosocysteine (SNOC), a NO donor that triggers the release of endogenous 5028

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Chart 7. Structures of 53−64

bright regions in the TPM image overlapped well with the OPM image due to LTR, indicating that the former reflected the [Zn2+]mito distribution. Moreover, the TPEF intensity increased upon treatment with 2,2′-dithiodipyridine (DTDP),133 a reagent that promotes the release of Zn2+ from Zn2+-binding proteins, and decreased when carbonyl cyanide m-chlorophenylhydrazone (CCCP),134 a compound that promotes the release of intramitochondrial cations by collapsing the mitochondrial membrane potential, was added. This outcome established that 62 can detect the changes in the [Zn2+]mito in the cells. The utility of 62 in tissue imaging was also established.132 To simultaneously detect cytosolic free Zn2+ ([Zn2+]cyto) and [Zn2+]mito, a set of two TP probes that emit TPEF at different wavelength ranges was developed (63 and 64).135 BZn-Cyto (63) and FZn-Mito (64) are derived using BODAN and 7(benzo[d]thiazol-2-yl)-9,9-dimethyl-9H-fluorene (BMF) as the fluorophores and DPEN derivatives as the Zn2+ chelators. Compounds 63 and 64 are TP turn-on probes that have been shown to have TPFEF values of 27 and 16, as well as KdTP values of 1.8 and 17 nM, respectively, and the intensity of their emission is increased in the presence of excess Zn2+. These probes showed high selectivity for Zn2+ and pH-insensitivity in the biologically relevant pH range. Moreover, the emission maximum of 64 was red-shifted 90 nm from that of 63. These

Interestingly, this spectral shift has been attributed to the enhanced electron-accepting ability and/or planarity that occur upon complexation, thereby increasing the ICT. This Zn2+mediated shift in the emission of this probe has been exploited for the detection of this ion using TPM in live cells and tissues, including 60-labeled HeLa cells and rat hepatocytes.130 To detect Zn2+ in mitochondria ([Zn2+]mito), TP turn-on probes 61 and 62, derived using 6-(benzo[d]thiazole-2′-yl)-2(N,N-dimethylamino)naphthalene (BTDAN) as the fluorophore, DPEN as the Zn2+ chelator, and TPP as the mitochondria-targeting moiety, were developed (Chart 7).131,132 Compounds 61 and 62 showed fluorescence maxima at 500 and 536 nm, respectively. The red-shifted emission observed for 62 is likely due to the stronger electronwithdrawing ability of the benzothiazole moiety in 62 than of that in 61. Probes 61 and 62 showed TPFEF values of 7 and 68 and KdTP values of 3.1 and 1.4 nM, respectively. The enhanced TPFEF and reduced KdTP values for 62 have been attributed to the 2-OMe group in the receptor moiety, as was demonstrated for 54 (Table 7). 61 and 62 respectively showed Φδmax values of 75 and 155 GM in the presence of excess Zn2+, high selectivity for Zn2+, and pH-insensitivity in the biologically relevant pH range. The utility of 62 in cell imaging was tested by a colocalization experiment with HeLa cells colabeled with 62 and MTR. The 5029

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Table 7. Photophysical Data for 53−64 probe 53, AZn1 53−Zn2+ 54, AZn2 54−Zn2+ 55, AZnM2 55−Zn2+ 56, AZnE2 56−Zn2+ 57, DZn 57−Zn2+ 58, AD2 58−Zn2+ 59, 6-MPVQ 59−Zn2+ 60, GBC 60−Zn2+ 61, SZn-Mito 61−Zn2+ 62, SZn2-Mito 62−Zn2+ 63, BZn-Cyto 63−Zn2+ 64, FZn-Mito 64−Zn2+

solvent h

H2O

H2Oh H2Oj H2Ok H2Ol H2Om MeOH/H2On MeCN/H2Or H2Oo H2Op H2Oq H2Oq

a λ(1) max

365 365 365 365 364 363 366 364 403 403 374 411 320 320 403 430 388 375 413 395 367 nas 392 nas

λflmaxb

Φc

496 498 494 499 504 504 503 504 610 610 546 570 412 493 530 610 500 493 536 536 470 nas 559 nas

0.022 0.47 0.012 0.65 0.015 0.42 0.026 0.39 0.020 0.62 0.034 0.016 0.036 0.266 0.89 0.60 0.15 0.92 0.0048 0.33 0.025 nas 0.023 nas

d λ(2) max i

nd 780 ndi 780 ndi 780 ndi 780 810 810 800 840 710 720 800 815 ndi 750 ndi 750 740 740 nas 800

δe i

nd 210 ndi 140 ndi 268 ndi 226 400 935 133 205 335 470 107 1433 ndi 82 ndi 470 160 nas nas nas

Φδf i

nd 86 ndi 95 ndi 110 ndi 86 8 580 4.5 3.3 12 125 95 860 ndi 75 ndi 155 4 103 nas 80

Kdg 1.1 nM (1.1 nM) 0.5 nM (0.5 nM) 8.4 nM (7.1 nM) 23 nM (21 nM) 0.51 μM (0.52 μM) 20.3 nM 0.45 nM 10 μM 3.1 nM (3.1 nM) 1.4 nM (1.4 nM) 1.8 nM 17 nM

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gKd values measured by one-photon mode. The values in parentheses are measured by two-photon mode. hData were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM EGTA, pH 7.2) in the absence and presence (1.8 μM) of Zn2+. iNot determined. The TPEF intensity was too weak for the cross section to be measured accurately. jData were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM NTA, pH 7.3) in the absence and presence (1.3 μM) of Zn2+. kData were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM NTA, pH 7.3) in the absence and presence (0.46 μM) of Zn2+. lData were measured in 30 mM MOPS (100 mM KCl, 10 mM EGTA, pH 7.2) in the absence and presence (6 μM) of Zn2+. mData were measured in 10 mM HEPES buffer (0.1 M NaCl, pH 7.4) in the absence and presence (10 μM) of Zn2+. nData were measured methanol− water solutions (1:1, v/v, 50 mM HEPES buffer, pH 7.4) in the absence and presence (40 μM) of Zn2+. oData were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM EGTA, pH 7.2) in the absence and presence (120 nM) of Zn2+. pData were measured in 50 mM HEPES buffer (100 mM KCl, 10 mM NTA, pH 7.4) in the absence and presence (47 nM) of Zn2+. qData were measured 30 mM MOPS buffer (100 mM KCl, pH 7.2) in the absence and presence of excess Zn2+. rData were measured in acetonitrile−H2O (1:1 v/v HEPES buffer, pH 7.2) in the absence and presence (60 μM) of Zn2+. sNot available. a

Φδmax values in the presence of excess Hg2+ (Table 8).140,141 They also showed pH insensitivity over the biologically relevant pH range, low cytotoxicity, and high photostability. The utility of 65 and 66 for the detection of Hg2+ in fish organs was tested. Fishes were reared for 1 and 3 days in aquaria containing 2 ppb of HgCl2 according to Organization for Economic Co-operation and Development (OECD) guidelines.142 Fishes were euthanized, and the organs were dissected and stained with 65. TPM images of the organs labeled with 65 showed that the Hg2+ concentration was highest in the kidney, followed by heart, liver, and gills (Figure 11). The Hg2+ concentration estimated by TPM in the kidney after 3 day exposure was 40 ppb, a value very similar to the 42 ppb measured by inductively coupled plasma mass spectrometry (ICPMS).140 These results demonstrated that 65 can detect [Hg2+] in fresh fish organs by TPM at a much lower concentration than 0.55 ppm, the upper level of Hg2+ in edible fish permitted by the United States Environmental Protection Agency (US EPA) standard.143 Compounds 67−69 are reaction-based TP probes for Hg2+. 67 is based on Hg2+-induced desulfurization of the probe molecule followed by opening of the spirolactam ring (Chart 8).144 Upon addition of excess Hg2+, the TPEF intensity of 67

properties allowed for dual-color imaging of [Zn2+]cyto and [Zn2+]mito in live cells and tissues.135 7.6. Two-Photon Probes for Mercury, Cadmium, Lead, and Nickel Ions

Heavy-metal ions in the environment can accumulate in fish or animals, be transported to humans through the food chain, and cause disease.89−91 Mercury is the most toxic heavy-metal ion in all its forms, including elemental, inorganic, and organic.136 Cadmium and lead are also toxic, as they can replace calcium or zinc at the active site in various proteins, leading to formation of complexes with cellular thiol species.137,138 The toxicity of nickel is relatively low compared with other heavy metals, but its accumulation can cause lethal effects in aquatic ecosystems.139 To understand heavy-metal-related concerns, it is important to develop TP probes that can detect heavy-metal ions in living organisms. To this end, a variety of TP probes for Hg2+, Cd2+, Pb2+, and Ni2+ have been developed (65−74, Chart 8). Compounds 65 and 66 are TP turn-on probes for Hg2+ derived from use of acedan as the fluorophore and azathiocrown ether as the Hg2+ chelator (Chart 8). Notably, these probes are highly selective for Hg2+ and have TPFEF values of 6 and 60, KdTP values of 0.45 and 0.51 μM, and large 5030

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To detect Cd2+ in live cells, two TP probes have been developed (70, 71). Compound 70 is a turn-on probe derived using acedan as the fluorophore and N,N,N′,N′-tetra[(2hydroxyethyl)carbamoylmethyl]-o-phenylenediamine as the Cd2+ chelator (Chart 8).147 The TPFEF value for this probe was determined to be 27, with a Kd value of 72 μM and a large Φδmax value in the presence of excess Cd2+ (Table 8). The utility of 70 to detect Cd2+ in live HepG2 cells was also investigated.147 Another example is the ratiometric TP probe derived from 6-styrylquinoline (71, Chart 8),148 which showed red-shifted emission maxima from 475 to 520 nm upon complexation with Cd2+ in MeOH/H2O (1:9). This outcome has been attributed to the enhanced ICT created by the coordination of the quinoline nitrogen with the Cd2+ ion. The Cd2+ Kd value for 71 was 2.72 nM, and the spectral intensity increased by 7-fold upon complexation with Cd2+ (Table 8). The TPM-imaging experiments of 71-labeled HeLa cells showed that this probe is able to monitor Cd2+ influx in living cells. Compound 72 is a TP turn-off probe for Pb2+ derived using dicyanostilbene as the fluorophore and N,N-bis(2aminophenylthioethyl)aniline as the Pb2+ receptor.149 The TPEF intensity of 72 decreased in response to Pb2+, with Kd values of 0.76 μM (Table 8). The Φδmax value of 72 in MOPS buffer was 540 GM at 790 nm. The utility of 72 in live cells and mouse brain tissue slices has been reported.149 Compounds 73 and 74 are TP turn-on probes for Ni2+ derived from use of acedan as the fluorophore and N,N-bis[2(carboxylmethyl)thioethyl]amine as the Ni2+ chelator (Chart 8).150 The TPFEF values of 73 and 74 were 5 and 26, respectively. The enhanced TPFEF of 74 was attributed to the prolinamide linker, as reported for ANa2 (40). The Kd value of 73 for Ni2+ was 100 μM, which was similar to that of 74 (Table 8). Both probes showed high selectivity for Ni2+, large Φδmax values, low cytotoxicity, high photostability, and pH insensitivity over a biologically relevant pH range. The utility of 74−AM in detecting Ni2+ in fish organs was tested. The TPM images of fresh fish organs labeled with 74− AM showed that [Ni2+] in fish organs decreased in the order kidney > heart > gill ≥ liver, a result consistent with that

Figure 10. TPM images of a rat hippocampal slice stained with 54. (a) TPM image at a depth of ∼120 μm with 10× magnification. (b) Magnification at 100× in the stratum lucidum (SL) of CA3 regions (yellow box) at a depth of ∼100 μm. (c, d) TPM images in the hilus (H) of dentate gyrus (DG) regions at a depth of ∼100 μm (c) before and (d) after addition of KCl to the imaging solution. Scale bars: (a, c) 300 μm and (b) 150 μm. Modified with permission from ref 122. Copyright 2008 Wiley-VCH.

increased by 20-fold in MeCN/H2O (1:1), presumably because the ring-opening reaction produced the rhodamine moiety (Chart 8). A significant Φδmax value was also observed for 67 in the presence of excess Hg2+ in HEPES buffer (Table 8). The utility of 67 to detect Hg2+ in living HeLa cells was also investigated.144 Compounds 68 and 69 are based on the Hg2+catalyzed deprotection of thioacetal and ketal. The TPM imaging applications of 68 and 69 in live cells have been reported.145,146 Chart 8. Structures of 65−74

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Table 8. Photophysical Data for 65−74 probe 65, AHg1 65−Hg2+ 66, AHg2 66−Hg2+ 67 67−Hg2+ 68, DTA1 68−Hg2+ 69, SAN 69−Hg2+ 70, TPCd 70−Cd+ 71, APQ 71−Cd2+ 72, DPb 72−Pb2+ 73, ANi1 73−Ni2+ 74, ANi2 74−Ni2+

solvent h

H2O

H2Oj H2Ol H2Om H2On H2Oo H2Op H2Oq H2Or H2Or

a λ(1) max

361 362 361 361 535 535 311 369 nak 352 365 365 340 340 400 400 365 365 375 375

λflmaxb 489 498 476 476 545 545 428 525 nak 503 502 502 475 520 609 609 500 500 500 500

Φc 0.025 0.15 0.0073 nak nak nak nak nak nak nak 0.035 0.453 0.057 0.2124 0.53 nak 0.017 0.088 0.0080 0.21

d λ(2) max i

nd 780 ndi 750 ndi 780 nak nak nak nak 740 740 720 710 790 790 ndi 750 ndi 750

δe i

nd 846 ndi nak ndi nak nak nak nak nak 102 242 206 400 1020 380 ndi 364 ndi 429

Φδf i

nd 110 ndi 78 ndi 55 nak nak nak nak 3.6 110 12 85 541 nak ndi 32 ndi 90

Kdg 0.46 μM (0.45 μM) 5.0 μM (5.1 μM) nak nak nak 61 μM (72 μM) 2.72 nM 0.764 μM (0.758 μM) 100 μM (100 μM) 88 μM (89 μM)

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gKd values measured by one-photon mode. The values in parentheses are measured by two-photon mode. hData were measured in 20 mM HEPES buffer (pH 7.0) in the absence and presence (2.35 μM) of Hg2+. iNot determined. The two-photon excited fluorescence intensity was too weak for the cross section to be measured accurately. jData were measured in 20 mM HEPES buffer (pH 7.0) in the absence and presence (60 μM) of Hg2+. kNot available. lData were measured in acetonitrile−water solution (1:1, v/v, 20 mM HEPES buffer, pH 7.0) in the absence and presence (2 μM) of Hg2+. m Data were measured in acetonitrile−water solution (1:99, v/v, 10 mM HEPES buffer, pH 7.4) in the absence and presence (20 μM) of Hg2+. nData were measured in PBS buffer (pH 7.4) in the absence and presence (10 μM) of Hg2+. oData were measured in 20 mM Tris-HCl buffer (0.1 mM sodium phosphate, pH 7.4) in the absence and presence (0.8 mM) of Cd2+. pData were measured in methanol−water solution (1:9, v/v, 50 mM HEPES buffer, pH 7.4) in the absence and presence (37.5 μM) of Cd2+. qData were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM EGTA, pH 7.2) in the absence and presence (40 μM) of Pb2+. rData were measured in 20 mM HEPES buffer (pH 7.1) in the absence and presence (10 μM) of Ni2+. a

reported for [Hg2+]. Moreover, a linear relationship was found between the TPEF intensities and [Ni2+] as measured by ICPMS. These results confirmed that 74 is capable of detecting Ni2+ in fish organs.150

These results indicate that 75 and 76 are suitable to detect acidic regions in cells and tissues by TPM. Indeed, the TPM images of 76-labeled macrophages displayed bright spots, which were found to be acidic vesicles. The utility of 76 to detect acidic vesicles in fresh rat hippocampal tissue has been established.155 Compound 77 is a ratiometric TP probe suitable to detect neutral pH (Chart 9)156 that was derived from use of 2diethylamino-7-(benzo[d]thiazol-2-yl)-9H-fluorene as the reporter, 2-diethylamino group as the protonation site, and two propionic acid groups at the 9,9-position as the watersolubilizing group. The fluorescence spectra of 77 increased gradually at 391 nm with a concomitant decrease at 570 nm upon acidification, presumably because of the shortened conjugation length caused by the protonation at the amino group. Interestingly, the measured pKa value of 77 is 6.95, and the Φδmax value decreased dramatically upon protonation (Table 9). The TPM image of 77-labeled NT2 cells revealed that this probe distributed well in the cytosol.156 Two ratiometric TP probes for pH derived using D−π−A dipoles having p-methoxyphenyl (78) or 2-methoxynaphthyl (79) as the donor, oxazole as the conjugation bridge, and pyridine as the acceptor and protonation sites were reported (Chart 9).157,158 As the pH was changed from 8 to 3, the emission maxima of 78 showed a gradual red-shift from 465 to 530 nm, presumably because of the enhanced ICT from the donor to the acceptor upon protonation at the pyridyl nitrogen. The pKa value of 78 was 5.7 in Britton−Robinson buffer, and

8. TWO-PHOTON PROBES FOR pH Intracellular pH (pHi) is an essential factor that controls many cellular metabolic processes, such as signaling, endocytosis, apoptosis, and proliferation.151−153 The pHi varies from 4.0 to 8.0, depending upon the specific subcellular compartment.151 While the pH in the cytosol is tightly regulated to near 7.2, some organelles, such as lysosomes (pH 4.0−5.5) and endosomes (pH 4.5−6.8), have acidic pH values, which is linked to enzyme activity and protein degradation.56,154 Abnormal pHi levels underlie certain cellular dysfunctions, which can cause many diseases, including cancer and neurodegenerative disorders. To understand the roles of pHi, it is crucial to monitor the pHi at the cell, tissue, and organismal levels. For this purpose, a variety of TP probes for pH detection have been developed (75−83, Chart 9). Compounds 75 and 76 are TP turn-on probes for pH derived using acedan as the fluorophore and aniline as the protonation site (Chart 9).155 The TPEF intensities of 75 and 76 increased gradually upon acidification, with TPFEF values of 26 (75) and 68 (76). The larger TPFEF value for 76 has been attributed to the 2-OMe group in the aniline moiety, as explained for the AZn series (53 and 54). Both compounds have a pKa value of 4.5 as well as large Φδmax values (Table 9). 5032

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Fgreen [measured for 79 at 400−500 nm (Fblue) and 600−750 nm (Fgreen) in universal buffer], versus pH was a sigmoidal curve. The pKa value of 79, estimated from this curve, was 4.4, a value smaller than that of 78, as expected from the absence of an electron-donating 2-amino group. This outcome suggests that the pHi can be estimated by using this curve and the Fblue/ Fgreen ratio as measured in the 79-labeled cells and tissues. Moreover, the Φδmax value for 79 was 3-fold larger than that of 78, a discrepancy that has been attributed to the extended conjugation length present in 79 (Table 9). These results indicate that 79 is a useful TP probe for pH. Indeed, the ratiometric TPM images constructed from the Fblue/Fgreen ratio measured in 79-labeled human tissue were found to be an effective tool for estimating the pH values of the ex vivo slices of human stomach and esophagus (see section 15, “TwoPhoton Probes for Medical Applications”). Recently, a series of benzimidazole-based ratiometric TP probes (80−83) for acidic pH was developed (Chart 9).159 Compounds 80−82 showed a marked blue-to-green emission color change in response to pH variation, presumably because of the enhanced ICT upon protonation at the benzimidazolyl nitrogen (Table 9). The pKa value of 80 was 5.89, which varied from 4.88 to 6.10 depending upon the substituents in the benzimidazole moiety (Table 9). Furthermore, the Φδmax values were shown to vary from 80 to 82 at pH 3.5 and, as a result of the enhance ICT observed for the protonated form, were 3.5-fold larger than those measured at pH 7.2 (Table 9). Probe 83, which has the tertiary amino group as the protonation site, was developed as a lysosome-targeted probe (see section 5, “Two-Photon Probes for Lysosomes and Mitochondria”). The spectral properties of 83 were almost identical to those of 80, except that the Φδmax value was larger by 2-fold. The ratiometric TPM imaging studies revealed that 80 is suitable to estimate the pH in both acidic and neutral regions throughout the subcellular compartments of a cell. Moreover, the changes of lysosomal pH in 83-labeled HeLa cells could be

Figure 11. (a, upper row) TPM images of 65-labeled organs of Oryzias latipes obtained at 100 μm depth by magnification at 10× (a, lower row). The regions indicated by the red boxes in the upper row are magnified 100×. Scale bar: 300 μm (upper) and 30 μm (below). (b) Relative TPEF intensity of 65-labeled organs after exposure to 2 ppb Hg2+ for 1 and 3 days. The TPEF was collected at 500−620 nm upon excitation with femtosecond pulses at 780 nm. Modified with permission from ref 140. Copyright 2010 Royal Society of Chemistry.

the spectral intensity increased by more than 3-fold upon protonation (Table 9). The larger Φδmax value for 78−H+ was attributed to the enhanced ICT. A similar result was observed for 79 with a blue-to-green shift in the emission maxima upon acidification.158 The plot of the emission intensity ratios, Fblue/ Chart 9. Structures of 75−83

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Table 9. Photophysical Data for 75−83 probe 75, AH1 76, AH2 77 78, PYMPON 79, NP1 80, BH1 81, BH2 82, BH3 83, BH1L

pH h

3.2 7.0h 3.2h 7.0h 4.0j 10j 3.0k (2.0) 8.0k (9.0) 2.5h 6.5h 3.5h 7.2h 3.5h 7.2h 3.5h 7.2h 3.5h 7.2h

a λ(1) max

λflmaxb

Φc

d λ(2) max

δe

Φδf

pKag

364 365 365 365 341 382 355 326 398 342 368 337 370 339 365 345 368 341

498 498 496 496 391 570 530 465 630 500 494 455 499 457 488 451 490 448

0.60 0.03 0.64 0.01 0.21 0.56 0.70 0.80 0.021 0.084 0.76 1.00 0.76 1.00 0.79 0.93 0.72 1.00

780 ndi 780 ndi 680 770 710 710 740l

140 ndi 138 ndi 1 100 60 15 155l

86 ndi 88 ndi 0.21 56 42 12 155l

4.40 (4.50) 4.50 (4.50) 6.95

750 740 750 740 750 750 750 750

185 40 200 50 80 20 390 95

140 40 155 50 65 20 280 95

6.90 4.36 5.91 (5.89) 4.92 (4.88) 6.11 (6.10) 5.82 (5.86)

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gpKa values measured by one-photon mode. The values in parentheses are measured by two-photon mode. hData were measured in universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl). iNot determined. The two-photon excited fluorescence intensity was too weak for the cross section to be measured accurately. jData were measured in aqueous buffer. kData were measured in Britton−Robinson buffer. The pH values in parentheses are conditions of the two-photon mode. lThe value of two-photon cross section measured in DMF. The fluorescence quantum yield of NP1 is 1.00 in DMF. a

Figure 12. (a) Merged image of ratiometric TPM images of 83-labeled HeLa cells with the corresponding DIC image. (b, c) Enlarged ratiometric TPM images of the white box in part a showing the changes of pH values before and 3.5 min after addition of 5 mM NH4Cl to the imaging solution. (d) Ratiometric TPM images of a rat hippocampal slice stained with 83. A total of 120 TPM images along the z-direction at depths of approximately 90−180 μm were accumulated to visualize the overall pH distribution with 10× magnification. (e) Higher-magnification images (63×) of the DG region. Modified with permission from ref 159. Copyright 2013 American Chemical Society.

9. TWO-PHOTON PROBES FOR THIOLS AND HYDROGEN SULFIDE

estimated in almost real-time (Figure 12). To further demonstrate the utility of 83 in mapping pH values in live tissues, ratiometric TPM images were obtained from a 83labeled rat hippocampal slice at a depth of 90−180 μm. The accumulated TPM image revealed that the acidic region was more widespread in the DG region than in the CA regions (Figure 12).

Amino acids and peptides containing thiol groups including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are components of many polypeptides and play vital roles in biology.160−162 They control redox homeostasis through the equilibrium between thiols (RSH) and disulfides (RSSR) and 5034

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Chart 10. Structures of 84−94

(86) as the fluorophores and aldehyde as the reaction site for the thiols (Chart 10). In CH3CN/Tris-HCl buffer (4:1, pH 7.3), 85 showed high selectivity for Cys over other amino acids, a TPFEF value of 11, and an emission red-shifted by 84 nm in response to Cys. This result has been attributed to the formation of two thiazolidine rings in 85, which altered the probe symmetry from A−D−A pseudo-octupole to D−π−A dipole. A similar result was observed for 86. Furthermore, both compounds were found to have large δmax values when evaluated in MeCN/buffer (4/1) and MeOH/buffer (1/1) (Table 10). The utility of 85 and 86 in the TPM imaging of live HeLa cells has been reported.168 Compound 87 is a TP probe for thiols derived from use of acedan as the fluorophore and a disulfide group as the reaction site for the thiols (Chart 10).169 The reaction between 87 and 2-aminoethanethiol (2-AET) followed second-order kinetics with k2 = 2.2 × 10−2 M−1 s−1, indicating that the reaction proceeded by the rate-limiting attack of thiols at the disulfide bond followed by the cleavage of the C−N bond to yield acedan (Chart 10). The TPFEF value of the reaction was 10 and the Φδmax value calculated for the reaction product was 10fold larger than that of unreacted 87 (Table 10). The TPM images of HeLa cells labeled with 87 were bright. The TPEF intensity increased after treatment with α-lipoic acid, which increases GSH production,170 and decreased upon addition of N-ethylmaleimide (NEM), a thiol blocking agent,171 indicating the utility of 87 in detecting thiols in live cells. Colocalization experiments with 87 and MTR revealed that the thiols exist predominately in the mitochondria. The utility of 87 in live tissue imaging was also demonstrated.169

maintain the higher-order structures of proteins.163 Abnormal level of thiols can cause cardiovascular and neurological disorders. In mitochondria, GSH exists mainly in the reduced form, with the ratio of reduced to oxidized form (GSH:GSSG) greater than 100:1. An increased GSH:GSSG ratio is considered to be a hallmark of oxidative stress.164 Hydrogen sulfide (H2S) is a signaling molecule that has diverse functions, including modulation of redox status, neuroprotection from oxidative stress, and anti-inflammation.165,166 In the brain, a defect in H2S production is related to neurodegenerative diseases. To understand the roles of thiols and H2S in biology, it is crucial to monitor their levels in live cells and tissues. For this purpose, a variety of TP probes for thiols (84−89) and H2S (90−94) have been developed (Chart 10). The first example of a TP probe for thiols was 8-oxoacenaphthopyrrole (84),167 which readily reacted with 3thiopropionic acid in MeOH, forming the adduct via the SNArH mechanism (Chart 10). The color of the reaction mixture changed from yellow-green to red-orange with a concomitant increase in the fluorescence intensity that can be attributed to the enhanced ICT in the adduct. 84 showed a high selectivity for cysteine (Cys) and homocysteine (Hcy) over other amino acids; a FEF value of 75; and a Φδmax value of 10 GM at 1000 nm for the adduct in MeOH/HEPES buffer (7:3, pH 7) (Table 10). The utility of 84 in TPM imaging of live and fixed cells has been reported.167 Compounds 85 and 86 utilize the formation of thiazolidine rings by the reaction between aldehyde and β-aminothiols for their sensing mechanism.168 They were derived using 4vinylpyridine-substituted triphenylamine (85) and carbazole 5035

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Table 10. Photophysical Data for 84−94 probe 84, AC1 84−Cys,Hcy 85, AM1 85−Cys 86, CA1 86−Cys 87, ASS 87-(2-AET) 88 88-GSH 89, SSH-Mito 89−GSH 90, FS1 90−H2S 91, NHS1 91−H2S 92, TFCA 92−H2S 93, SHS-M1 93−H2S 94, SHS-M2 94−H2S

a λ(1) max

solvent g

MeOH/H2O

MeCN/H2Oi MeOH/H2Ok H2Ol H2Om H2On H2Oo DMF/H2Op H2Or H2Os H2Os

430 580 375 375j 360 410 321 382 nah 434 338 383 352 363 338 358 360 363 340 365 343 383

λflmaxb 588 588 493 577 nah 540 457 503 nah 514 462 545 529 548 nah 480 444 548 420 500 464 545

Φc

d λ(2) max

δe

Φδf

0.008 nah nah nah nah nah 0.22 0.17 nah 0.34 0.82 0.12 0.025 0.46 nah nah 0.013 0.46 0.23 0.50 0.24 0.12

h

h

nah nah nah nah nah nah 11 113 nah 276 80 55 15 302 1.85q 111q 0.67 52 14 63 17 55

na 1000 nah 920 nah 810 780 780 nah 860 740 750 750 750 740p 760p 740 750 750 750 740 750

na 11 nah 1700 nah 90 50 665 nah 814 95 550 600 657 nah nah 52 113 61 126 71 458

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gData were measured in methanol−water solution (7:3, v/v, HEPES buffer, pH 7.0) before and 10 min after addition (200−330 μM) of homocysteine or cysteine. hNot available. iData were measured in acetonitrile−water solution (4:1, v/v, 10 mM Tris/HCl buffer, 100 mM KCl, pH 7.3) before and after addition (600 μM) of cysteine. jA new absorption band is generated between 450 and 550 nm. kData were measured in methanol−water solution (4:1, v/v, 10 mM Tris/HCl buffer, 100 mM KCl, pH 7.0) before and after addition (600 μM) of cysteine. lData were measured in 30 mM MOPS buffer (100 mM KCl, 10 mM EGTA, pH 7.2) before and 2 h after addition (10 mM) of 2-aminoethanethiol. mData were measured in PBS buffer (pH 11, 1% EtOH). nData were measured in 30 mM MOPS buffer (100 mM KCl, pH 7.4) before and 2 h after addition (10 mM) of glutathione. oData were measured in 20 mM HEPES buffer (100 mM KCl, pH 7.2) before and 2 h after addition (100 μM) of Na2S. pData were measured in DMF−water solution (8:2, v/v, 20 mM HEPES buffer, pH 7.4) before and 60 min after addition (100 μM) of NaHS. qData were measured in DMF−water solution (1:1, v/v, 20 mM HEPES buffer, pH 7.4). rData were measured in 20 mM HEPES buffer (100 mM KCl, pH 7.4) before and 2 h after addition (100 μM) of Na2S. sData were measured in 30 mM HEPES buffer (100 mM KCl, pH 7.4) before and 1 h after addition (100 μM) of Na2S. a

Φδmax value was also observed after the probe reacted with GSH (Table 10). The emission intensities ratio, Fyellow/Fblue, measured at 425−475 nm (Fblue) and at 525−575 nm (Fyellow), increased by 45-fold in the presence of 10 mM GSH. The colocalization experiment of 89-labeled HeLa cells with MTR revealed that 89 predominantly localizes in mitochondria. Upon 740 nm excitation, the ratio Fyellow/Fblue of 89-labeled HeLa cells increased from 1.24 to 2.64 after treatment with αlipoic acid and decreased to 0.77 upon addition of NEM (Figure 13), indicating the utility of 89 in monitoring mitochondrial thiols in living cells. The TPM image of fresh hippocampal slices labeled with 89 displayed the distribution of the thiols (Figure 13). The changes in the ratio as measured in fresh hippocampal slices were comparable to those obtained in the cells. To detect hydrogen sulfide (H2S), a TP turn-on probe (90) derived using BMF as the TP reporter and azide as the H2S reaction site was developed.176 This probe reacted with Na2S (100 μM) in HEPES buffer (pH 7.2) with the k2 = 2.9 M−1 s−1, which was attributed to the rate-limiting attack by the thiolate ion followed by the decomposition of the resulting adduct to an amino group.177 The TPFEF value was 21, and the TP-induced brightness measured for the reaction product increased 20-fold compared to that of unreacted 90 (Table 10). The TPM images of HeLa cells labeled with 90 were bright, as expected from the

Compound 88 is a TP turn-on probe for thiols. This probe was designed by employing a rigid analogue of the chromophore green fluorescent protein (GFP) as the fluorophore, and S-phenyl carbonothioate as the reaction site for thiols.172 Probe 88 has a TPFEF value of 30 with a k2 = 4.4 × 10−2 M−1 s−1 and is highly selective for thiols (GSH, cysteine, and homocysteine) over other biorelevant species. Furthermore, this probe also has a large Φδmax value (Table 10). The utility of 88 to detect H2S in live HeLa cells and in live mouse liver tissue has been reported.172 In the mitochondria, thiols (including GSH) play a crucial role in controlling the redox status and in repairing oxidative damage.173,174 To detect mitochondrial thiols, a reaction-based ratiometric TP probe (89) was developed (Chart 10).175 This probe consists of three components: BTDAN as the TP reporter, a disulfide bond as the thiol reaction site, and TPP as the mitochondria-targeting moiety. The disulfide bond and TPP were separated as far as possible to minimize possible interactions between them. Upon addition of GSH to 89 in MOPS buffer (pH 7.4), the emission spectra increased gradually at 545 nm with a concomitant decrease at 462 nm. The k2 value determined for this reaction was 2.3 × 10−2 M−1 s−1, a value nearly identical to that measured for 87. Notably, this probe was highly selective for thiols (over other amino acids without thiol groups), metal ions, and H2O2. A significant 5036

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Figure 13. Images of a rat hippocampal slice stained with 89. (a, d) Bright-field images of the CA1−CA3 regions. (b, e) Ratiometric TPM images of a fresh rat hippocampal slice (b) nontreated and (e) pretreated with NEM before labeling with SSH-Mito. Ten ratiometric TPM images were accumulated along the z-direction at depths of approximately 90−190 μm with 10× magnification. (c, f) Enlarged images of the red box in parts b and e, respectively. Scale bars: 300 μm (a, d) and 75 μm (c, f). Modified with permission from ref 175. Copyright 2011 American Chemical Society.

large Φδmax value of the reaction product. The TPEF intensity increased after treatment with Cys or GSH, which are the precursors to H2S production,177,178 and decreased when the cells were stimulated with phorbol myristate acetate (PMA), which is known to decrease endogenous H2S level by inducing phagocytosis-associated ROS generation,179 indicating the utility of 90 in detecting H2S in live cells. The utility of 90 in live tissue imaging was also demonstrated.176 Subsequently, a BTDAN-based TP turn-on probe (91) for H2S was developed that has an azide as the reaction site.180 Probe 91 showed an FEF value of 67 and high selectivity for NaHS over other reactive sulfur, oxygen, and nitrogen species, as well as over anions, and was pH-insensitive in the biologically relevant pH range. The Φδmax value was also observed to increase by 50-fold upon completion of the reaction (Table 10). The utility of 91 to detect H2S in live HeLa cells was reported.180 The sulfide-induced deazidation reaction has been successfully utilized for the quantitative detection of H2S levels. TFCA (92) is a ratiometric TP probe designed by introducing an azide-based carbamate leaving group to 7-(benzo[d]thiazol-2yl)-9,9-(2-methoxyethoxy)ethyl-9H-fluorene-2-amine (TFA) as the TP fluorophore.181 Upon addition of 100 μM Na2S to 92 in HEPES buffer (pH 7.4), the fluorescence intensity increased gradually at 548 nm with a concomitant decrease at 444 nm. The k2 value determined for this reaction in HEPES/EtOH was 5.17 M−1 s−1, which is nearly identical to that measured in HEPES buffer. The emission intensity ratio of 92, F2/F1, measured at 390−450 nm (F1) and 525−650 nm (F2), increased by 110-fold in the presence of 100 μM Na2S, with a detection limit of 0.086 μM. This probe displayed a high selectivity for sulfides over other biologically relevant species and demonstrated pH insensitivity. Notably, the spectral intensity of the reaction product was increased 8-fold compared to that of unreacted 92 (Table 10).

Ratiometric TPM images using 92 revealed the sulfide levels in fresh rat colon tissues (Figure 14). A kinetic method was also developed to quantitatively estimate the total sulfide concentration ([H2S] + [HS−]) in live cells and tissue.181 The kobs values were determined by the kinetic method, and the total sulfide concentration was calculated by using the equation kobs = k2[HS−], where k2 = 5.17 M−1 s−1 and [H2S]/[HS−] = 0.794. The total sulfide concentration was found to be almost zero in live HeLa cells and 4−7 μM in fresh rat colon tissue (Figure 14). To quantitatively detect H2S levels in mitochondria, ratiometric TP probes (93 and 94) derived using BTDAN as a TP fluorophore, 4-azidobenzyl carbamate as the H2S reaction site, and TPP as the mitochondrial targeting unit were developed.182 The emission maxima of 93 and 94 were 420 and 464 nm, respectively, in 30 mM HEPES buffer (pH 7.4) (Table 10). The red-shifted emission for 94 is due to the stronger electron-withdrawing ability of the 6-carbamoyl group in 94 compared with that of the 6-alkoxy group in 93. When 94 was treated with 100 μM Na2S in HEPES buffer, the emission intensity increased gradually at 545 nm with a concomitant decrease at 464 nm. A similar result was observed for 93, except that the two emission maxima were significantly blue-shifted. The k2 values for the reactions of 93 and 94 with Na2S were 5.8 and 7.0 M−1 s−1, respectively. These probes showed a high ratiometric response for Na2S over other reactive sulfur, oxygen, and nitrogen species. The relative brightness of the reaction products was also large for both probes (Table 10). The colocalized TPM images of 94-labeled HeLa cells with MTR revealed that 94 exists predominantly in mitochondria.182 Upon treatment of the 94-labeled cells with GSH or Cys, which are precursors to H2S, the Fyellow/Fblue ratio measured at 425− 470 nm (Fblue) and 525−575 nm (Fyellow) increased from 0.49 to 0.76 or 0.81, respectively. A similar result was observed in ratiometric TPM images of 94-labeled fresh rat hippocampal 5037

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Figure 14. Pseudocolored ratiometric TPM images of mouse colon tissue labeled with 92. (a) TPM images of tissue before (upper column) and after (bottom column) treatment with 5-fluorouracil at a depth of about 90−170 μm with magnification at 20×. Scale bar: 150 μm. (b) 3D images (top, tilted, and side views) of the colon tissues constructed from 150 TPM images obtained before (left column) and after (right column) treatment with 5-fluorouracil. The white box shows an expanded image of the selected region (white dotted box) with magnification at 100×. (c) A figure showing mucosa, muscularis mucosae, and submucosa layers. (d) F2/F1 ratios measured 2 h after incubation with 92 in the mucosa and submucosa before and after treatment with 5-fluorouracil. (e) Sulfide concentration in mucosa and submucosa before and after treatment with 5-fluorouracil estimated by the kinetic method. Modified with permission from ref 181. Copyright 2013 American Chemical Society.

in living organisms.189 It plays a vital role in synaptic activity in the brain and in immune system defense, whereas misregulation of NO production is implicated in cancer and neurodegenerative disorders.190,191 To understand the roles of ROS and RNS in biology, it is crucial to monitor their levels in live cells and tissues. For this purpose, a number of TP probes for H2O2 (95, 96), O2•− (97), ROS (98, 99), and NO (100, 101) have been developed (Chart 11). Compound 95 is a reaction-based TP probe derived using acedan as the fluorophore and aryl boronate as the H2O2 reaction site, which are linked together through the carbamate group (Chart 11).192 Upon treatment of 95 with 1 mM H2O2 in HEPES buffer (20 mM, pH 7.1), the fluorescence at 500 nm increased gradually with a concomitant decrease at 450 nm, with a kobs value of 1.0 × 10−3 s−1, presumably because the H2O2-mediated conversion of boronate to phenol followed by cleavage of the carbamate linkage liberated the amino group (Chart 11). Probe 95 displayed high selectivity for H2O2 over other competing ROS and RNS molecules. The emission intensities ratio Fgreen/Fblue, measured at 390−465 nm (Fblue) and 500−550 nm (Fgreen), increased by 10-fold in the presence of 1 mM H2O2, with a concomitant increase in brightness greater than 3-fold (Table 11). The Fgreen/Fblue ratio of 95labeled Raw 264.7 cells increased from 0.89 to 1.28 upon addition of phorbol myristate acetate (PMA), a reagent that

slices. These outcomes established the utility of 94 for the detection of endogenous H2S levels in living cells and live tissue. Notably, ratiometric TPM imaging using 94 was found to be an effective tool for measuring different H2S levels in normal tissue and in Parkinson’s disease model systems (see section 15, “Two-Photon Probes for Medical Applications”).

10. TWO-PHOTON PROBES FOR REACTIVE OXYGEN SPECIES (ROS) AND REACTIVE NITROGEN SPECIES (RNS) ROS and RNS are important mediators in a variety of physiological and pathological processes. The prominent members of the ROS family include hydrogen peroxide (H2O2) and the superoxide anion radical (O2•−).183,184 While the precise flow of H2O2 is crucial for the normal cell functions such as cell signaling and defense, overexposure to H2O2 is implicated in many human diseases. 185 The abnormal generation or accumulation of H2O2 in mitochondria has also been linked to cancer and Alzheimer’s and Parkinson’s diseases.186,187 Also, the misregulated production of O2•−, most of which occurs in mitochondria, can cause cell death because it can be readily transformed into H2O2 and other ROS.188 Nitric oxide (NO), the archetype of the RNS family, modulates a variety of physiological and pathological processes 5038

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Chart 11. Structures of 95−101

Table 11. Photophysical Data for 95−101 probe

solvent

a λ(1) max

λflmaxb

Φc

d λ(2) max

δe

Φδf

95, PN1 95−H2O2 96, SHP-Mito 96−H2O2 97, MF-DBZH 97−O2•‑ 98 98−MeO 99 99−MeO 100, ANO 100−NO 101, QNO 101−NO

H2Og

321 338 342 383 naj naj 452 452 491 490 370 370 408 408

453 500 470 545 512 512 473 474 511 540 502 502 535 535

0.70 0.40 0.13 0.12 naj 0.10k 0.04 0.95 0.14 0.92 0.009 0.70 0.016 0.20

720 740, 750 740 750 naj naj naj 670 naj 970 ndo 750 ndo 810

17 113 85 458 naj 13.7k naj 500 naj 400 ndo 243 ndo 260

12 45 11 55 naj 1.37k naj 475 naj 368 ndo 170 ndo 52

H2Oh H2Oi CHXl H2Om H2On H2Op

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gData were measured in 20 mM HEPES buffer (pH 7.1) before and 1 h after addition (1 mM) of H2O2 (hydrogen peroxide). hData were measured in 30 mM MOPS buffer (100 mM KCl, pH 7.4) before and 1 h after addition (1 mM) of H2O2. iData were measured in 10 mM Tris-HCl (pH 8.0) before and after addition (12 μM) of superoxide anion. jNot available. kData were measured in 10 mM Tris-HCl (pH 7.4) after addition (40 μM) of superoxide anion. lData were measured in cyclohexane. mData were measured in water (pH 8.0). nData were measured in 30 mM PBS buffer (100 mM KCl, pH 7.4) before and 15 min after addition (100 μM) of NO (nitric oxide). oNot determined. The two-photon excited fluorescence intensity was too weak for the cross section to be measured accurately. pData were measured in 100 mM PBS buffer (containing 5% CH3CN, pH 7.4) before and after addition (100 μM) of NO. a

induces H2O2 generation through a cellular inflamation response. A similar result was observed after treatment with 100 μM H2O2. The ability of 95 to monitor the H2O2 level in live tissue has also been demonstrated.192

To detect mitochondrial H2O2, a TP probe (96) derived using BTDAN as the fluorophore, aryl boronate-based carbamate leaving group as the H2O2 reaction site, and TPP as the mitochondria-targeting moiety was developed (Chart 5039

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11).193 Upon addition of 1 mM H2O2 to 96 in MOPS buffer (30 mM, pH 7.4), the emission intensity at 545 nm increased, with a concomitant decrease at 470 nm; the kobs value was 1.1 × 10−3 s−1. The Fyellow/Fblue value, measured at 400−470 nm (Fblue) and 530−600 nm (Fyellow), increased 75-fold upon completion of the reaction, and the detection limit of H2O2 was 4.6 μM. This probe exhibited high selectivity for H2O2 over other competing ROS and RNS molecules, as well as a 5-fold increase in spectral intensity upon completion of the reaction (Table 11). The colocalized TPM images of Raw 264.7 murine macrophage cells colabeled with 96 and MTR confirmed that 96 is predominantly localized in mitochondria. Moreover, the Fyellow/Fblue ratio of 96-labeled macrophages increased from 0.62 to 1.62 and 1.56 after treatment with H2O2 and PMA, respectively. The TPM image of fresh hippocampal slices labeled with 96 revealed the distribution of H2O2 in different regions of the tissue. Furthermore, the changes in the Fyellow/ Fblue ratio measured in the fresh hippocampal slices were comparable to those determined in the cells. These results established the utility of 96 in detecting mitochondrial H2S in living cells and live tissue.193 To detect mitochondrial O2•−, a TP probe (97) derived from use of fluorene as the TP reporter, dibenzothiazoline group as the O2•− reaction site, and TPP as the mitochondria-targeting moiety was developed (Chart 11).194 This probe utilized O2•−mediated oxidation of the benzothiazoline moiety as its detection mechanism. Probe 97 showed a 3.5-fold turn-on response in the presence of O2•− in Tris-HCl buffer (10 mM, pH 8.0); high selectivity for O2•− over ROS, RNS, GSH, and ascorbic acid molecules; high photostability; pH insensitivity; and low cytotoxicity (Table 11). The detection limit of 97 for O2•− was 9.5 nM, and the Φδmax value of the reaction product of 97 was 1.37 GM (Table 11). A colocalization experiment with HepG2 cells colabeled with 97 and Mito-SOX Red established that 97 is predominantly localized in mitochondria. The TPEF intensity in the 97-labeled HepG2 cells increased after stimulation with PMA and decreased upon treatment with a Tiron solution (a cell permeable O2•− scavenger), indicating the utility of 97 in monitoring mitochondrial O2•− in live cells. The TPM images of mouse abdomen tissue incubated with 97, acquired at the depth of 150−250 μm, were brighter when treated with lipopolysaccharide (LPS) than were those of untreated control tissue, indicating the utility of 97 in tissue imaging (Figure 15). Profluorescent TP probes (98 and 99) for ROS derived using anthracene and fluorescein as the fluorophores and nitroxyl radial as the ROS reaction site were developed (Chart 11).195 Compounds 98 and 99 emitted little fluorescence due to quenching of the excited state by the paramagnetic nitroxide. Upon reaction with ROS, they were rapidly converted to the diamagnetic and nonquenching species, making them sensitive probes for ROS. The fluorescence quantum yields of the nonradical model compounds (98−MeO and 99−MeO) were much higher than those of 98 and 99. Further, the large Φ values associated with these model compounds resulted in their Φδmax values also being very large (Table 11). The utility of 98 and 99 for imaging H2O2-induced oxidative stress in live cells has also been established by TPM.195 To detect NO, a TP turn-on probe (100) derived using acedan as the fluorophore and o-diaminobenzene as the NO reaction site was developed (Chart 11).196 Upon reaction with NO, the o-diaminobenzene moiety of 100 was converted to benzotriazole, with a concomitant 68-fold increase in the TPEF

Figure 15. TPM images of mice with an LPS-mediated abdomen injury. (A) O2•− was produced inside the peritoneal cavity of the mice during the LPS-mediated inflammatory response. (B) In situ TPM images of mouse abdomen incubated with 97. Images were acquired using 770 nm TP excitation. The TP fluorescence emission window was 500−550 nm. Modified with permission from ref 194. Copyright 2013 American Chemical Society.

intensity. Probe 100 showed a fast response (within 5 min) to NO; high selectivity for NO over ROS, RNS, ascorbic acid, and dehydroascorbic acid molecules; pH insensitivity; low cytotoxicity; and high photostability. The detection limit was 5 nM and the Φδmax value of the reaction product was 170 GM at 750 nm (Table 11). The TPEF intensity in the 100-labeled cells increased upon treatment with LPS and interferon-γ (IFN-γ), well-known external activators that promote NO production by inducible nitric oxide synthase (iNOS), and was suppressed by the addition of L-NG-nitroarginine (L-NNA), an iNOS inhibitor. Similarly, the TPEF intensity of the 100-labeled tissue increased upon addition of N-methyl- D -aspartic acid (NMDA), a reagent that promotes NO production by cNOS (constitutive nitric oxide sythase), and was suppressed by the addition of N-(G)-nitro-L-arginine methyl ester (L-NAME), a cNOS inhibitor (Figure 16). These results confirmed the utility of 100 for detecting NO in live cells and tissues. Subsequently, a TP turn-on probe for NO (101) was developed by employing the same strategy as above, except that quinoline was used as a TP reporter (Chart 11).197 Probe 101 showed an FEF value of 12 upon reaction with NO, kobs = 8.25 × 10−3 s−1, high selectivity for NO, pH insensitivity, low cytotoxicity, and high photostability. The detection limit of 101 for NO was 0.084 μM and the Φδmax value of the reaction product was 52 GM at 810 nm (Table 11). TPM imaging experiments with 101 showed that this probe can detect endogenous NO produced in live cells and living tissue at a depth of 90−180 μm.197

11. TWO-PHOTON PROBES FOR GLUCOSE AND ATP Glucose is widely used as the primary energy source for cell growth in an organism. The transport of glucose into the cell, a process called glucose uptake, occurs by the action of glucose transporters (GLUT1−4) and by passive diffusion.198,199 Glucose uptake is quicker in fast-growing cancer cells than in normal cells.200 While the insulin-mediated signaling pathway stimulates the glucose uptake in cells and tissues, a defect in insulin secretion or action can lead to diabetes, metabolic syndrome, hypertension, and cardiovascular diseases.201,202 After uptake, the oxidation of glucose to CO2 and water produces adenosine triphosphate (ATP), which stores the energy needed for cellular activity. ATP and other biophosphates are implicated in numerous cellular events, such as energy metabolism, signaling, and DNA replication.203,204 5040

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Figure 16. (a) TPM Images of DG regions in a rat hippocampal slice stained with 100 before and after addition of 1 mM NMDA or 100 mM LNAME and 1 mM NMDA to the imaging solution. (b) Time course of the TPEF intensity in part a. The images were collected at 425−575 nm upon excitation at 750 nm with a femtosecond pulse. Scale bar: 75 μm. Modified with permission from ref 196. Copyright 2012 Wiley-VCH.

Chart 12. Structures of 102−105

Table 12. Photophysical Data for 102−105 probe 102, AG1 103, AG2 104, AS1 104−D-glucose 105 105−ATP

solvent h

H2O H2Oh H2Oj H2Oj H2Ol H2Ol

a λ(1) max

373 375 366 365 410 400

λflmaxb 501 501 500 498 558 538

Φc 0.90 0.56 0.040 0.16 0.02 0.10

d λ(2) max

780 780 ndk 780 nai nai

δe 95 155 ndk 531 nai nai

Φδf 86 88 ndk 85 nai nai

Kdg i

na nai 0.60 M (0.62 M) 0.16 μM

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gKd values measured by one-photon mode. The values in parentheses are measured by two-photon mode. hData were measured in 30 mM PBS buffer (pH 7.4). iNot available. jData were measured in 30 mM PBS buffer (pH 7.4) in the absence and presence (1.0 M) of D-glucose. kNot determined. The two-photon excited fluorescence intensity was too weak for the cross section to be measured accurately. lData were measured in 10 mM HEPES buffer (pH 7.4, containing 1% MeCN) in the absence and presence (1.0 M) of D-glucose. a

Φδmax values of 86 and 88 GM at 780 nm, respectively (Table 12), pH insensitivity in the biologically relevant pH range, negligible cytotoxicity, and high photostability under the TPM imaging conditions. TPM images of 103-labeled cells revealed that the uptake of 103 was faster in cells derived from cancer than in normal cells. Probe 103 competed with D-glucose for cellular uptake, but not with L-glucose or osmotic pressure, indicating that the uptake occurs through a glucose-specific transport system rather than

Deficiencies in ATP levels are directly linked to diseases including ischemia, hypoglycemia, and neuronal disorders.205 To monitor glucose uptake and detect ATP in live cells and tissues by TPM, various TP tracers and TP turn-on probes have been developed (102−105, Chart 12). Probes 102 and 103 are TP tracers developed to monitor glucose uptake.206 They were designed by connecting α-Dglucose to acedan through a 3,6-dioxaoctane-1,8-diamine (102) or piperazine (103) linker. Compounds 102 and 103 showed 5041

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Figure 17. TPM images of 104-labeled primary cortical neurons at (a) 100 s and (b) 300 s. (c) Changes in the TPEF intensity of 104-labeled primary cortical neurons as a function of time. D-Glucose, D-fructose, and D-galactose (20 mM) were added after 200 s. TPM images of 104-labeled fresh rat hippocampal slices at a depth of ca. 100 μm obtained at (d) 100 s and (e) 1500 s. After 200 s, the tissue was treated with D-glucose, insulin, and KCl. (f) Time course of TPEF intensity at designated positions 1−3 in part d. The TPM images were collected at 500−620 nm upon excitation at 780 nm with femtosecond pulses. Scale bars: (a) 30 μm and (d) 300 μm. Modified with permission from ref 207. Copyright 2012 Royal Society of Chemistry.

Chart 13. Structures of 106−112

intensity of 104 increased gradually upon addition of D-glucose with a TPFEF value of 4 in a PBS buffer solution (10 mM, pH 7.4) and a Kd value of 0.60 M (Table 12). For comparison, the Kd values of 104 for D-galactose and D-fructose were 0.35 and 0.048 M, respectively. Moreover, the Φδmax value of the 104−Dglucose complex was 85 GM at 780 nm (Table 12). Probe 104 showed negligible cytotoxicity and high photostability under the TPM imaging conditions. The TPM images of 104-labeled HeLa cells and primary cortical neurons showed dramatic increases in TPEF intensity upon addition of D-glucose. In contrast, no change was observed with addition of D-galactose and D-fructose (Figure 17). This result confirmed the utility of 104 as a TP probe for glucose. A similar result was observed in

by passive diffusion. These results established the utility of 103 as a TP glucose tracer. Moreover, when A549 cells were treated with taxol or combretastatin, well-known anticancer drugs, the uptake rate slowed down, in both a time- and dose-dependent manner.206 The uptake rate of 103 was faster in cancer tissue than in normal tissue and showed a remarkable decrease upon treatment with taxol. Here again, the effect was dosedependent. These results suggest that 103 may find useful application in the diagnosis of colon cancer and in screening of anticancer agents (see section 15, “Two-Photon Probes for Medical Applications”). Compound 104 is a TP turn-on probe for glucose derived from use of acedan as the fluorophore and a boronic acid moiety as the glucose-binding unit (Chart 12).207 The TPEF 5042

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Table 13. Photophysical Data for 106−112 probe 106, D-pepep 106−DNA 107, TP-2py 107−DNA 108, TP-3py 108−DNA 109, S-carbazole 109−DNA 110, V-carbazole 110−DNA 111, 9E-BHVC 111−DNA 112, 2,7-9E-BHVC 112−DNA

solvent g

H2O

H2Oi H2Oi H2Ol H2Ol H2Om H2On

a λ(1) max h

na nah 474, 491j 509 474, 491j 499 416 418 436 451 451 474 434 450

λflmaxb

Φc

d λ(2) max

δe

Φδf

600 nah 656j 665, 658k 665j 684, 683k 573 563 573 548 566 540 567 565

0.10 0.40 0.11j 0.035, 0.066k 0.12j 0.021, 0.013k 0.02 0.07 0.0029 0.13 0.0011 0.39 0.0004 0.01

707 nah 820j 820k 820j 820k 850 880 830 810 800 800 800 830

347 nah 315j 200k 700j 700k 431 3127 1241 2170 2200 230 4172 4422

34.7 nah 34.7j 13.2k 84j 9.1k 8.6 218 3.6 284 2.4 89.5 1.67 44.2

λmax of the one-photon absorption spectra in nm. bλmax of the one-photon emission spectra in nm. cFluorescence quantum yield. dλmax of the twophoton excitation spectra in nm. eThe peak two-photon cross section in 10−50 cm4 s/photon (GM units). fTwo-photon action cross section. gData were measured in PBS buffer (pH 7.0) in the absence and presence of calf thymus DNA (ct-DNA). hNot available. iData were measured in 10 mM sodium cacodylate buffer (100 mM NaCl, pH 7.0) in the absence and presence (25 μM) of oligonucleotide ds26. jData were measured in glycerol. k Data were measured in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of Herring testes DNA. DNA (base pairs)/dye ratio: 150 for (TP-2 py) and 25 for (TP-3 py), respectively. lData were measured in PBS buffer (pH 7.0) in the absence and presence (3−120 μM) of ct-DNA. m Data were measured in 10 mM Tris-HCl buffer (100 mM KCl, pH 7.2) in the absence and presence of ct-DNA. Values of phosphate of DNA/dye ratio: 70. nData were measured in Tris-HCl buffer in the absence and presence of DNA. Nucleic acid (NA)/dye ratio: 100. a

Compound 106 is the first example of a TP probe for DNA and is derived using pyrrole as the electron donor, pyridinium ion as the electron acceptor, and the CC bonds as the conjugation bridge (Chart 13).213 Probe 106 showed an emission maximum at 600 nm in PBS buffer (pH 7.0) as well as a high affinity for DNA, with a Kd value of 0.5 μM, an FEF value of 4, low cytotoxicity, and a modest Φδmax value (Table 13). The utility of 106 was demonstrated by the brighter TPM image of 106-labeled Saccharomyces cerevisiae cells than those labeled with DAPI, a well-known OP fluorescent probe for DNA.7 Compounds 107 and 108 were developed using a similar strategy as described above, except that triphenylamine was used as the donor (Chart 13).214,215 Upon binding with DNA, 107 and 108 displayed emission maxima at 658 and 683 nm, respectively, and a remarkable turn-on response, with Kd values in the micromolar range. The fluorescence enhancement was attributed to inhibition of the rotational relaxation processes due to the immobilization of the probes in the DNA matrix.214 Similar results were observed in glycerol, which was employed as a model for the highly viscous medium surrounding the DNA matrix. The Φδmax values for 107 and 108 in glycerol were 35 and 84 GM at 820 nm, respectively (Table 13). A colocalization experiment with CHO-K1 cells colabeled with 108 and DAPI revealed that this probe can selectively detect the DNA in the nucleus. The TPM images of 108-labeled CHO-K1 cells clearly displayed the chromosomes in the nuclei during cell divisions (Figure 18). Compounds 109−112 were developed as above using carbazole as the electron donor (Chart 13).216−218 Probes 109 and 110 showed fluorescence maxima at 573 and 578 nm in PBS buffer (pH 7.0), respectively. Upon binding with DNA, they showed spectral blue-shifts with significantly enhanced Φ (Table 13). The larger FEF values observed in AT-rich doublestranded DNA (dsDNA) versus GC-rich dsDNA indicated the selective binding of these probes with electronegative AT-rich sites.216 Upon binding with DNA, the brightness of the emission spectra increased by 70-fold. A similar result was also

TPM images of 104-labeled fresh rat hippocampal slices (Figure 17).207 Compound 105 is a TP turn-on probe for ATP and ADP (Chart 12) derived using acedan as the fluorophore and a 2,2′dipicolylamine−Zn2+ complex as the ATP- and ADP-binding motif.208 The emission spectrum of 105 was red-shifted by 50 nm from that of acedan in the presence of Zn2+, presumably because of the increased acceptor ability of the acyl group upon coordination with Zn2+. The Kd values of 105 for ATP and ADP were 0.16 and 3.1 μM, respectively, with FEF values of 7− 9 (Table 12). Probe 105 showed high selectivity toward ATP and ADP over other biological anions, including AMP and pyrophosphate (PPi), presumably because of the favorable π−π interactions between the acedan core and the adenine base of ATP and ADP (Chart 12). The TPM images of 105-labeled cells upon excitation at 740 nm indicated the utility of 105 in live cells.208

12. TWO-PHOTON PROBES FOR NUCLEIC ACIDS Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are biomacromolecules essential for all life. DNA has a doublehelical structure composed of double-stranded (ds) polynucleotides and carries the genetic information needed for the production of proteins and functional RNAs in the cells.209 Damage to the DNA can cause mutagenesis, carcinogenesis, aging, and neurodegenerative disorders.210−212 To visualize DNA at the subcellular level by TPM, TP probes derived from a D−A dipole (109), A−D−A quadrupoles (106, 107, 110− 112), and an octupole (108) possessing pyrrole (106), triphenylamine (107, 108), and carbazole (109−112) as the electron donor and pyridinium ion as the electron acceptor been developed (Chart 13). These nucleic acid probes utilize the electrostatic interaction between cationic groups in the probe and electronegative sites in DNA (such as on phosphate), so they can be bound to and emit enhanced TPEF from the DNA matrix due to the restricted rotation of C−C bonds in the probe. 5043

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1,6-elimination. Labeling HeLa cells with 113 also revealed that the endogenous activities of PTP occur mostly in the cytosol. A similar labeling strategy has also been successfully extended to the development of another TP probe for cathepsin activity.224 Compound 114 is another TP turn-on probe that was derived using a phosphate as the reaction site, but instead it uses 2-hydroxy-4,6-bis(4-hydroxystyryl)pyrimidine as the TP fluorophore (Chart 14).225 Upon reacting this probe with phosphatase, the emission fluorescence was blue-shifted from 532 to 512 nm with a 4-fold increase in the brightness (Table 14). Furthermore, when fresh brain tissue isolated from 1-dayold female Drosophila was labeled with 114, the phosphate activity was observed to occur mostly in the medulla region (Figure 19). Moreover, introduction of different cell-penetrating peptides (CCPs) onto the 114 probe allowed organelleand tumor-cell-specific imaging of the phosphatase activity occurring in these cellular regions (Chart 14). Probes 115 and 116 were also developed to investigate enzyme-mediated reactions, specifically those performed by monoamine oxidases (MAOs). Compound 115 is a TP turn-on probe that was designed on the basis of the intramolecular cyclization reaction.226 Prior to treatment with MAO, 115 emitted minimal levels of fluorescence, a phenomenon that is likely due to the efficient PeT from the amino group to the fluorophore. However, upon treatment with MAOs, the fluorescence of this probe increased abruptly as a result of the enzymatic oxidation of the amino group to an iminium ion, followed by β-elimination and intramolecular cyclization generating the reaction product, as shown in Chart 14. Notably, the TPM images of 115-labeled chromaffin cells (MAOs expressed cell line) were much brighter than those of C6 glioma cells (MAOs not expressed cell line). Compound 116 was developed by employing acedan as the TP reporter and 3-aminopropyl carbamate as the MAO-Bresponsive unit (Chart 14).227 Probe 116 showed an emission maximum at 449 nm in HEPES buffer as well as a red-shifted and enhanced fluorescence (498 nm, Φ = 0.19) in the presence of MAO-B (Table 14). The Michaelis−Menten constant (Km) determined by kinetic assay of MAO-B/116 was 8.33 μM. Moreover, the TP brightenss was increased by more than 7-fold upon completion of the reaction (Table 14). This allowed the detection of the MAO-B level in live cells and tissues by TPM. The utility of this probe for detecting MAO-B activity in a PD model was demonstrated (see section 15, “Two-Photon Probes for Medical Applications”). Compound 117 is a TP turn-on probe that has been designed specifically to measure tyrosinase activity. It was derived using a phenol as the tyrosinase reaction site and acedan as the TP reporter, the two of which are linked through a urea moiety.228 In the presence of tyrosinase, a turn-on signal was observed as a result of phenol oxidation followed by the liberation of the 5-amino-o-benzoquinone and acedan. Notably, the use of this probe for TPM imaging of 117-labeled live cells has revealed that tyrosinase-expressing cells, such as B16-F1 cells, emitted a fluorescent signal that was much brighter than that observed for tyrosinase-deficient cells, such as HeLa cells, highlighting the specificity of this probe for this enzyme. Cyclooxygenase-2 (COX-2) is another enzyme that has been targeted using TP probes (118 and 119). Interestingly, these TP probes respond to enzyme binding by undergoing a conformational change. To target this COX-2, 118 utilizes acenaphtho[1,2-b]quinoxaline (ANQ) as the TP fluorophore, indomethacin (IMC) as the COX-2 recognition site, and

Figure 18. (A) One-photon and (B, D) two-photon microscopy images of 108-labeled CHO-K1 cells. (C) Transmission image corresponding to part D. The excitation wavelengths were (A) 543 and (B, D) 840 nm. Modified with permission from ref 214. Copyright 2007 Wiley-VCH.

observed for 109 (Table 13). The utility of 110 was tested using TPM images of HeLa cells labeled with 110.216 Probes 111 and 112 showed similar spectral changes upon binding with DNA, except that after binding with DNA, the spectral intensity for both probes was 2−6-fold weaker (Table 13). The utility of 111 and 112 in TPM imaging experiments has also been established.217,218

13. TWO-PHOTON PROBES FOR ENZYMES Enzymes are efficient catalysts that accelerate the rates of biochemical reactions.219−222 In the human body, there are many different types of enzymes the activities of which are highly selective and site-specific. Furthermore, abnormal enzyme activity has been directly related to a number of diseases, such as neurodegenerative disorders, cancer, and aging.219−222 To visualize the activity of specific enzymes in situ, various TP probes have been developed to monitor numerous different enzyme-mediated biological reactions (113−120, Chart 14). For example, 113 and 114 are TP probes designed to detect phosphatase activity. Compound 113 is a TP turn-on probe based on the FRET from the fluorophore to the quencher (Chart 14).223 This probe was derived using a phosphate as the reaction site for protein tyrosine phosphatase (PTP), 4-(4dimethylaminophenyl)diazenylbenzoic acid as the quencher, and 2-hydroxy-4,6-bis(4-(diethylamino)styryl)pyrimidine as the TP fluorophore. To increase cell loading of this probe, the phosphate group was protected with a nitrobenzyloxy group, which can be easily removed with UV irradiation. Notably, when tested in HEPES buffer, 113 was only weakly fluorescent, with an emission maximum at 592 nm, indicating that the fluorescence is in fact being quenched by FRET. However, after irradiation with UV light and treatment with PTP, the TPEF intensity increased by 4-fold as a result of the nitobenzyloxy group being photocleaved and the quencher being removed by 5044

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Chart 14. Structures of 113−120

hexanediamine to link them together.229 In Tris-HCl buffer (pH 8.0), the efficient PeT occurring between the IMC and ANQ when the probe is in the folded state only permitted a weak fluorescent signal in both the one- and two-photon modes. However, upon binding with COX-2, an intense fluorescent signal was emitted. This outcome has been attributed to the unfolding of 118 upon binding with COX-2, limiting the level of PeT. Colocalization experiments using 118 and BODIPY TR C5 ceramide, a well-known marker for the Golgi apparatus, confirmed that 118, and thus COX-2, exists predominantly in this organelle. Moreover, TPM images of different cell lines labeled with 118 also revealed that COX-2 is more highly expressed in cancer cell lines compared to normal, noncancer cell lines. In fact, similar results were also observed in TPM images obtained of normal and sarcoma-180 liver slices labeled with 118, whereby increased COX-2 levels were observed in samples of the latter. Subsequently, 119, a nitro derivative of 118, was developed in order to fine-tune COX-2 detection (Chart 14).230 For example, at low COX-2 concentrations (