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We have developed blue- and yellow-emitting two-photon probes (BGolgi-blue and PGolgi-yellow) from 6-(benzo[ d]oxazol-2-yl)-2-naphthalylamine and 2,5-bis(benzo[ d]oxazol-2-yl)pyrazine derivatives as the fluorophores and trans-Golgi-network peptide (SDYQRL) as the Golgi-apparatus-targeting moiety. HeLa cells labeled with BGolgi-blue and PGolgi-yellow emitted two-photon-excited fluorescence at 462 and 560 nm, respectively, with effective two-photon-action cross-section values of 1860 and 1600 × 10

In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca

We report blue- and green-emitting two-photon probes derived from naphthalene and fluorene derivatives (as fluorophores) and an endoplasmic reticulum (ER) retrieval peptide (KDEL; as an ER-targeting moiety) that can detect the ER in a live cell by both one-photon and two-photon microscopy (TPM) and in a live tissue by TPM.

We have developed two-photon (TP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10–50cm4s/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl)/1,4-dioxane (7/3) solution. The TPM images of CCD-18co (a normal colon cell line) and HCT116 cells (a colon cancer cell line) labeled with BH-2 were too dim to be distinguished. When the same cells were labeled with BHEt-1, however, the TPM image of the HCT116 cells was much brighter than that of CCD-18co cells, and the relative proportion of the acidic vesicles (Pacid) of the former was 5-fold larger than that of latter. BHEt-1 could also differentiate HepG2 cells (a human liver cancer cell line) from LX-2 cells (a human hepatic stellate cell line) with a 6-fold larger Pacid value. Human colon cancer tissues labeled with BHEt-1 showed similar results, demonstrating...

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520-2830) × 10

We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 × 10–50 cm4s/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90–130 μm in live mouse tissue by TPM.

Two-photon microscopy (TPM) is a new imaging tool that can detect biological targets deep inside a live tissue. To faciltate the use of TPM in biomedical research, a variety of two-photon (TP) probes for specific applications are needed. In this Forum Article, we describe the design strategy, photophysical properties, and biological imaging applications of a selection of our recent studies in the development of TP probes for metal ions. Small-molecule TP turn-on probes, organelle-targeted probes, and multicolor emissive probes for dual-color imaging are briefly reviewed.

Hydrogen sulfide (H2S) is a multifunctional signaling molecule that exerts neuroprotective effects in oxidative stress. In this article, we report a mitochondria-localized two-photon probe, SHS-M2, that can be excited by 750 nm femtosecond pulses and employed for ratiometric detection of H2S in live astrocytes and living brain slices using two-photon microscopy (TPM). SHS-M2 shows bright two-photon-excited fluorescence and a marked change in emission color from blue to yellow in response to H2S, low cytotoxicity, easy loading, and minimum interference from other biologically relevant species including reactive sulfur, oxygen, and nitrogen species, thereby allowing quantitative analysis of H2S levels. Molecular TPM imaging with SHS-M2 in astrocytes revealed that there is a correlation between the ratiometric analysis and expression levels of cystathionine β-synthase (CBS), the major enzyme that catalyzes H2S production. In studies involving DJ-1, a Parkinson's disease (PD) gene, attenuated H2S production in comparison with wild-type controls was observed in DJ-1-knockout astrocytes and brain slices, where CBS expression was decreased. These findings demonstrate that reduced H2S levels in astrocytes may contribute to the development of PD and that SHS-M2 may be useful as a marker to detect a risk of neurodegenerative diseases, including PD.

Two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is a vital tool in biology and clinical science, due to its capacity to image deep inside intact tissues for a long period of time. To make TPM a more versatile tool in biomedical research, we have developed a variety of two-photon probes for specific applications. In this mini review, we will briefly discuss two-photon probes for lipid rafts, lysosomes, mitochondria, and pH, and their biomedical applications. [BMB Reports 2013; 46(4): 188-194]

We report two-photon probes (FMg1 and FMg2) that can selectively detect intracellular free Mg(2+) ([Mg(2+)](i)) in live cells and tissues by two-photon microscopy. Combined with BCaM, a two-photon probe for near-membrane Ca(2+) ([Ca(2+)](m)), FMg2 allows dual-color imaging of Mg(2+)/Ca(2+) activities in live cells and [Mg(2+)](i) /[Ca(2+)](m) distributions in live tissues at a depth of 100-200 μm.

Two-photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two-photon (TP) probes for specific applications are needed. In this article, we report a small-molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68-fold fluorescence enhancement in response to NO, and a maximum TP-action cross-section of 170 GM (GM: 10(-50) cm(4) photon(-1)) upon reaction with excess NO. This probe can be easily loaded into cells and tissues and can real-time monitor NO in living tissues at 100-180 μm depth for longer than 1200 s through the use of TPM, with minimum interference from other biologically relevant species.

pH values go live! A ratiometric two-photon (TP) probe (NP1, see scheme) that has a significant TP action cross-section, high photostability, negligible toxicity, and can estimate pH values in live cells and human tissues by two-photon microscopy is described. NP1 can detect the difference in pH between live cells from the gastroesophageal junction (GEJ) and the lower esophageal sphincter of patients with and without esophagitis.

We developed two-photon (TP) probes for DNA (ABI-Nu), cytoplasm (Pyr-CT) and mitochondria (BF-MT). We found that ABI-Nu binds to AT in the minor groove, while ABI-Nu and BF-MT are effective for tracking in the cytoplasm and mitochondria, respectively. These probes showed very large effective two-photon action cross section values of 2230, 1555 and 790 Göppert-Mayer units (1 GM = 10−50 cm4 s photon−1molecule−1) at 740 nm with emission maxima at 473, 561 and 560 nm, respectively, in each organelle. Using these probes, we quantitatively estimated the mean nuclear area and the ratios of nuclei to cytoplasm and mitochondria to nuclei in human colon tissues by dual-colour two-photon microscopy imaging within 2 h after biopsy. The mean nuclear area and the nuclei to cytoplasm and mitochondria to cytoplasm ratios increased in the following order: normal colon mucosa

We report two-photon Lysotrackers (CLT-blue and CLT-yellow) that can be excited by 750-840 nm femtosecond laser pulses and emit at 470 and 550 nm, respectively. They can be easily loaded into cells and tissue slices for visualization of lysosomes in live cells and tissues for a long period of time through two-photon microscopy. When combined with appropriate two-photon probes for other biological targets, these novel probes would greatly facilitate the two-photon microscopy colocalization experiments.

We report a ratiometric two-photon probe (SSH-Mito) for mitochondrial thiols. This probe shows a marked blue-to-yellow emission color change in response to RSH, a significant two-photon cross section, good mitochondrial thiol selectivity, low cytotoxicity, and insensitivity to pH over the biologically relevant pH range, allowing the direct visualization of RSH levels in live cells as well as in living tissues at 90-190 μm depth without interference from other biologically relevant species through the use of two-photon microscopy.

We report a two-photon (TP) probe (ACaLN) for near-membrane Ca(2+) that shows a 13-fold TP excited fluorescence (TPEF) enhancement in response to Ca(2+), dissociation constants (K(d)(TP)) of (1.9±0.2) μM, pH-insensitivity at the biologically relevant pH, and can detect near-membrane Ca(2+) in live cells for more than 1500 s and in living tissues at 120 μm depth without interference from other metal ions. Comparison with existing probes provides a useful strategy for the design of efficient TP probes for the near-membrane metal ions.

A series of Zn(2+)-selective two-photon fluorescent probes (AZnM1-AZnN) that had a wide range of dissociation constants (K(d) (TP) =8 nm-12 μM) were synthesized. These probes showed appreciable water solubility (>3 μM), cell permeability, high photostability, pH insensitivity at pH>7, significant two-photon action cross-sections (86-110 GM) upon complexation with Zn(2+), and can detect the Zn(2+) ions in HeLa cells and in living tissue slices of rat hippocampal at a depth of >80 μm without mistargeting and photobleaching problems. These probes can potentially find application in the detection of various amounts of Zn(2+) ions in live cells and intact tissues.

The lo domains, also called lipid rafts, are believed to serve as a platform to support various cellular processes such as signal transduction, membrane trafficking, pathogen invasion, cholesterol homeostasis, neurodegenerative diseases, and angiogenesis. The existence of lipid rafts has been demonstrated in mammalian cells and in model membranes by using various techniques such as atomic force microscopy, fluorescence microscopy, and coherent anti-Stokes Raman scattering microscopy. To understand their roles in cell biology, it is crucial to visualize such domains in the live cells and tissues. An ideal tool for this purpose is two-photon microscopy (TPM) that utilizes two near-infrared photons for excitation. The advantages of TPM include the capability of visualizing the real-time activities of the biological targets in live cells and intact tissues at >100 mm depth with minimum interference due to the tissue preparation artifacts that can extend ~70 mm from the tissue surface. For maximum utilization of TPM, many TP probes for specific applications are needed. However, TP probes for such applications are rare. Earlier, we reported a membrane TP polarity probe, CL, which showed several advantages over laurdan including greater sensitivity to the solvent polarity, brighter TPM image, and more precise reflection of the cell environment. We also developed a TP turn-on probe, CL2, which showed larger TP action cross-section and higher sensitivity to the polarity of the environment than CL. This allowed selective detection of the lipid rafts in live cells and tissues by TPM. Unfortunately, CL2 had one shortcoming; it internalized into the cytoplasm upon prolonged incubation with cells, causing a blurred TPM image. To overcome this problem, we have designed a new TP turn-on (off-on) probe (SL2) that has sodium sulfonate in the head group and exclusively stains the plasma membrane. Herein, we report that SL2 can detect the lipid rafts in the live cells and intact tissues without internalization problems by simply collecting the TP excited fluorescence (TPEF) by using TPM. The preparation of SL2 is given in the Supporting Information. The solubility of SL2 in water was 7 mm, which is sufficient to stain the cells (Figure S1 in the Supporting Information). The change of the head group from a carboxylic acid (CL2, 4 mm) to a sulfonate increased the water solubility by ~ twofold. The absorption and emission spectra of SL2 showed gradual bathochromic shifts with increasing solvent polarity (Figure 1A and Table S1) indicating the charge-transfer character of the emitting state. The shifts were larger for the emission than for the absorption spectra (21 vs. 126 nm). Moreover, the fluorescence quantum yield (F) increased by 22-fold as the solvent was changed from EtOH/H2O (F=0.04) to THF (F=0.88). CL2 showed similar behavior except that fluorescence enhancement factor (FEF) was lower [FEF(CL2)=16 versus FEF(SL2)= 22] (Table S1). Hence, the fluorescence of SL2 is more sensitive to the polarity of the environment than that of CL2. The fluorescence intensity of SL2 showed similar decrease with the hydrophilicity of the large unilamellar vesicles (LUVs) in the order, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (DPPC/CHL (40 mol%), lo)>DOPC/sphingomyelin/CHL (1:1:1, raft mixture)>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, ld ; Figure S2). The relative fluorescence intensities of SL2 in DPPC/CHL/raft mixture/DOPC in the 420–520 nm range are 7.0/4.8/1.0, respectively; SL2 emits sevenfold stronger fluorescence in the lo than in the ld domain. The slightly weaker fluorescence in the raft mixture than in DPPC/CHL can be explained if SL2 exists in 63/37 ratio in the lo/ld domains of the raft mixture with 7/1 intensity ratio (vide supra). A similar result was reported for CL2 (lo/ld=86:14). [12] This indicates high preference of these probes to reside in the lo domain, which can be attributed to the more favorable hydrophobic interactions with sphingomyelin when compared to DOPC. The sensitivity of the laurdan derivatives on the polarity of the environment originates from the intramolecular charge [a] C. S. Lim, J. H. Lee, Dr. Y. S. Tian, Prof. B. R. Cho Department of Chemistry, Korea University 1-Anamdong, Seoul, 136-701 (Korea) Fax: (+82)2-3290-3544 E-mail : chobr@korea.ac.kr [b] H. J. Kim, Prof. H. M. Kim Division of Energy Systems Research, Ajou University Suwon, 443-749 (Korea) E-mail : kimhm@ajou.ac.kr [c] Dr. C. H. Kim, Prof. T. Joo Department of Chemistry, Pohang University of Science and Technology Pohang, 790-784 (Korea) E-mail : thjoo@postech.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201000609.

The first example of cyclometalated platinum(II)-containing rhodamine probe (1) with two-photon induced luminescent properties was synthesized and investigated for mercury detection. A highly selective color change of 1, from light yellow to pink, is observed only in the presence of Hg2+ due to the formation of 1,3,4-oxadiazole ring in 2. This selectivity of Hg2+ with color changes can be observed easily by the naked-eye. Meanwhile, a remarkable turn-on and selective 20-fold fluorescent enhancement of 1 upon binding with Hg2+ over the other tested metal ions was observed. The water-soluble probe 1 was successfully applied in the visualizing of the site of Hg2+ accumulation as well as estimating of trace amounts of mercury ions in live HeLa cells by two-photon microscopy.

Two-photon fluorescent probes for the cellular membrane, derived from 6-acyl-2-aminonaphthalene as the fluorophore and hexanoyl (CH), lauryl (CL), and stearyl (CS) groups as the receptor, have been synthesized. Their photophysical properties and utility as membrane probes were also studied. Whereas CH cannot be used as a membrane probe due to its high water solubility, CL and CS are useful two-photon probes for membrane lateral heterogeneity, as they can easily stain cells, emit fluorescence with high sensitivity to the environment polarity, and are capable of imaging the membrane lateral heterogeneity in live cells. Moreover, CS is more likely to be located in the plasma membrane due to its negligible water solubility. Our results show that the liquid ordered-like domain covers 31-35% of the cellular surface.

The lipid-rafts hypothesis proposes that naturally occurring lipid aggregates exist in the plane of membrane that are involved in signal transduction, protein sorting, and membrane transport. To understand their roles in cell biology, a direct visualization of such domains in living cells is essential. For this purpose, 6-dodecanoyl-2-(dimethylamino)naphthalene (laurdan), a membrane probe that is sensitive to the polarity of the membrane, has often been used. We have synthesized and characterized 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), which has the advantages of greater sensitivity to the membrane polarity, a brighter two-photon fluorescence image, and reflecting the cell environment more accurately than laurdan. Lipid rafts can be visualized by two-photon microscopy by using C-laurdan as a probe. Our results show that the lipid rafts cover 38 % of the cell surface.

A novel, two-photon probe for the detection of free Mg2+ ions in living cells and live tissues has been developed. The probe can be excited by 880 nm laser photons, emits strong two-photon excited fluorescence in response to Mg2+ ions, can be easily loaded into the cell and tissue, shows high photostability, and can measure the Mg2+ ion concentration without interference by Ca2+ ions in living cells. The intracellular dissociation constant (Kdi) for Mg2+ determined by the two-photon process is 2.5 mM, which is suitable for dynamic Mg2+ concentration measurement. In addition, the probe is capable of imaging endogenous stores of free Mg2+ at a few hundred micrometers depth in live tissues using two-photon microscopy (TPM).

Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other.

We have developed TP probes for [Zn2+]cyto and [Zn2+]mito, which emit TPEF at widely-separated wavelength regions. The new probes can simultaneously detect [Zn2+]cyto and [Zn2+]mito in live cells, as well as in living tissues by dual-color TPM imaging.

Mitochondria provide the energy of the cells and are the primary site of oxygen consumption and the major source of reactive oxygen species. In mitochondria, metal ions and glutathione play vital roles in maintaining their structure and the redox environment. To understand their roles in mitochondria, it is crucial to monitor each of these chemical species in the mitochondria at the cell, tissue, and organism levels. An ideal tool for such purpose is the use of two-photon microscopy (TPM). Until recently, however, there has been no report on the two-photon (TP) probes suitable for such applications. In this paper, we summarize the mitochondria-targeted TP probes for Zn(2+), H(2)O(2), and thiols, as well as their bioimaging applications.

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts.

We report a two-photon fluorescent probe (SHP-Mito) which can ratiometrically detect mitochondrial H(2)O(2) in live cells and intact tissues at100 μm depth through the use of two-photon microscopy.

We report a two-photon turn-on probe (AS1) that can be excited by 780 nm femto-second pulses and visualize glucose uptake and the changes in the intracellular glucose concentration in live cells and tissue by two-photon microscopy.

Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observation of a single cell and molecules in intact tissues. Multiphoton microscopy exhibits nonlinear optical contrast properties, which can make it possible to provide an exceptionally large depth penetration with less phototoxicity. This system becomes more and more an inspiring tool for a non-invasive imaging system to realize "optical biopsy" and to examine the functions of living cells. In this review, we briefly present the physical principles and properties of multiphoton microscopy as well as the current applications in biological fields. In addition, we address what we see as the future potential of multiphoton microscopy for gastroenterologic research.

Molecular imaging by two-photon microscopy (TPM) has become indispensable to the study of biology/medicine owing to its capability of imaging deep inside intact tissues. To make TPM a more-versatile tool, a large variety of two-photon probes are needed. Herein, we report a new two-photon fluorescent probe (ANi2) that can be excited by 750 nm femtosecond pulses and detect Ni(2+) ions in fresh fish organs at 90-175 μm depth without interference from the pH value or from other biologically relevant species through the use of TPM. TPM images of fish organs labeled with ANi2 revealed that Ni(2+) ions accumulate in fish organs in the order: kidney > heart > gill ≥ liver. Moreover, a linear relationship was found between the two-photon-excited fluorescence (TPEF) and the inductively coupled plasma mass spectrometry intensities (ICP-MS), thereby allowing the quantitative measurement of Ni(2+) ions in live tissue.

We report a two-photon fluorescent probe (ACu1) that can be excited by 750 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize Cu(+) distribution in live cells and tissues by two-photon microscopy.

We report a two-photon probe (SZn-Mito) for mitochondrial zinc ions ([Zn2+]m). This probe shows a 7-fold enhancement of two-photon-excited fluorescence in response to Zn2+ with a dissociation constant (Kd(TP)) of 3.1 ± 0.1 nM and pH insensitivity in the biologically relevant range, allowing the detection of [Zn2+]m in a rat hippocampal slice at a depth of 100−200 μm without interference from other metal ions through the use of two-photon microscopy.

A naphthalimide-based highly selective colorimetric and ratiometric fluorescent probe for the fluoride ion displayed both one- and two-photon ratiometric changes. Upon reaction with the F(-) (TBA(+) and Na(+) salts) anion in CH(3)CN as well as in aqueous buffer solution, probe 1 shows dramatic color changes from colorless to jade-green and remarkable ratiometric fluorescence enhancements signals. These properties are mechanistically ascribed to a fluoride-triggered Si-O bond cleavage that resulted in a green fluorescent 4-amino-1,8-naphthalimide.

Two-photon microscopy (TPM) has become an indispensible tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. To make it a versatile tool in biology, a variety of two-photon probes for specific applications are needed. In this context, many research groups are developing two-photon probes for various applications. In this Focus Review, we summarize recent results on model studies and selected examples of two-photon probes that can detect intracellular free metal ions in live cells and tissues to provide a guideline for the design of useful two-photon probes for various in vivo imaging applications.

We report a two-photon fluorescent probe (AHg1) that can be excited by 780 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize the site of Hg(2+) accumulation, but can also estimate trace amounts of mercury ions in fresh fish organs by two-photon microscopy.

Sodium ion plays critical roles in many physiological and pathological processes such as nerve conduction, muscle and heart contraction; it modulates the electrolyte level, cation transport, and cell volume. [1] To understand these functions, a number of one-photon (OP) fluorescent probes have been developed, with SBFI and Sodium Green (SG) being commercially available (Figure 1). [2] A disadvantage of these probes is that they are bulky and suffer from difficulties in loading into live cells. The second problem, which is common for most OP probes, is their short excitation wavelength ( 500 μm), localized excitation, and prolonged observation time. [3] Using TPM, one can image intact tissue for a long period of time with minimum interference from tissue preparation artifacts that can extend more than 70 μm into the tissue slice. However, there is no report on the TP probe for Na+ that is capable of imaging [Na + ]i deep inside living tissues. To address both of these problems, we designed a two photon (TP) probe (ANa1) derived from 2acetyl-6-(dimethylamino)naphthalene (acedan) as the TP fluorophore and 1,7-diaza-15-crown-5 as the Na + ion receptor by considering the following requirements (Figure 1); (i) small MW for cell permeability; (ii) high selectivity for Na + ions; (iii) significant TP cross section for bright TPM image; (iv) large spectral shifts in different environments for discrimination between the cytosolic and membrane-bound probes; and (v) high photostability. Herein, we report that ANa1 is capable of detecting intracellular free Na + ions in live cells and tissues at >100 μm depth for a long period of

A two-photon fluorescent probe (ACaL) is reported that can be excited by 780 nm fs pulses, shows high photostability and negligible toxicity, and can visualize near-membrane Ca2+ in live cells and deep inside live tissues by two-photon microscopy.

Optical imaging with fluorescence microscopy is a vital tool in the study of living systems. The most common method for cell imaging, one-photon microscopy (OPM), uses a single photon of higher energy to excite the fluorophore. However, two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is growing in popularity among biologists because of several distinct advantages. Using TPM, researchers can image intact tissue for a long period of time with minimum interference from tissue preparation artifacts, self-absorption, autofluorescence, photobleaching, and photodamage. However, to make TPM a more versatile tool in biology, researchers need a wider variety of two-photon probes for specific applications. In this Account, we describe a series of two-photon probes that we developed that can visualize the distribution of intracellular metal ions, acidic vesicles, and lipid rafts in living cells and tissues. The development of these probes requires a significant two-photon cross section for the bright image and receptors (sensing moieties) that triggers the emission of the two-photon excited fluorescence upon binding with the ions or membrane in the living system. These probes also must be sensitive to the polarity of the environment to allow selective detection of cytosolic and membrane-bound probes. In addition, they need to be cell-permeable, water-soluble for the staining of cells and tissues, and highly photostable for long-term imaging. The resulting probes-AMg1 (Mg(2+)), ACa1-ACa3 (Ca(2+)), AZn1 and AZn2 (Zn(2+)), AH1, AH2, and AL1 (acidic vesicles), and CL2 (membrane)-use 2-acetyl-6-aminonaphthalene as the fluorophore and receptors for the target ions or membrane. All of these two-photon turn-on probes can detect the intracellular free metal ions, acidic vesicles, and lipid rafts at 100-300 microm depth in live tissues. Moreover, with ACa1-AM, we could simultaneously visualize the spontaneous Ca(2+) waves in the somas of neurons and astrocytes at approximately 120 microm depth in fresh hypothalamic slices for more than 1000 s without appreciable decay. Furthermore, AL1 could visualize the transport of the acidic vesicles between cell body and axon terminal along the axon in fresh rat hippocampal slices at approximately 120 microm depth.

We report a new two-photon fluorescence turn-on probe 6-[(E)-3-oxo-1-dodecenyl]-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (CL2) that is designed specifically for visualizing lipid rafts in living cells and tissues. This probe emits much brighter two-photon excited fluorescence in lipid rafts than in non-raft domains and allows direct visualization of the lipid rafts in the live cells and pyramidal neuron layer of the CA1 region at a depth of 100-250 mum in live tissues using two-photon microscopy.

2-Acetyl-6-(dimethylamino)naphthalene-derived two-photon fluorescent Ca2+ probes (ACa1-ACa3) are reported. They can be excited by a 780 nm laser beam, show 23-50-fold enhancement in one- and two-photon excited fluorescence in response to Ca2+, emit fourfold stronger two-photon excited fluorescence than Oregon Green 488 BAPTA-1 upon complexation with Ca2+, and can selectively detect intracellular free Ca2+ ions in live cells and living tissues with minimum interference from other metal ions and membrane-bound probes. Moreover, these probes are capable of monitoring calcium waves at a depth of 120-170 microm in live tissues for 1100-4000 s using two-photon microscopy with no artifacts of photobleaching.

We report a two-photon fluorescent probe (ASS) that can be excited by 780 nm femtosecond pulses and detect thiols in live cells and living tissues at a 90-180 microm depth without interference from other biologically relevant species by two-photon microscopy.

We report a highly sensitive two-photon probe (SZn2-Mito) which shows a 70-fold two-photon excited fluorescence enhancement in response to Zn(2+) and can selectively detect mitochondrial Zn(2+) in a rat hippocampal slice at a depth of 100-200 μm by using two-photon microscopy.

We report a two-photon fluorescent probe (PN1) that can be excited by 750 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize H(2)O(2) distribution in live cells and tissue by two-photon microscopy.