Yu Shun Tian

A two-photon fluorescent probe for thiols in live cells and tissues.

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.

Two-photon fluorescent probes for intracellular free zinc ions in living tissue.

Zinc is a vital component of enzymes and proteins. In the brain, a few millimoles of intracellular free Zn ions are stored in the presynaptic vesicles, released with synaptic activation, and seem to modulate excitatory neurotransmission. To understand the biological roles of Zn, a variety of fluorescent probes derived from quinoline (TSQ, Zinquin, and TFLZn) and fluorescein (FluZn-3, Znpyr, ZnAF, etc.) have been developed. However, most of them require a rather short excitation wavelength or suffer from pH sensitivity. To visualize the biological activity deep inside living tissue (> 80 mm) without the interference of surface preparation artifacts, it is crucial to use two-photon microscopy (TPM), which utilizes two photons of lower energy for the excitation. Recently, TPM has gained much interest from biologists because it offers a number of advantages in biological imaging, including increased penetration depth, localized excitation, and prolonged observation time. However, efficient two-photon (TP) probes for Zn appear to be rare. Furthermore, although a few pH-resistant sensors for Zn have been reported, they require either microinjection for cellular applications or use a significant amount of ethanol as co-solvent because of the poor water solubility. An efficient TP probe for Zn should have sufficient water solubility to stain the cells, high selectivity for Zn ions, significant TP cross section, pH resistance, and high photostability. In this context, we extend our earlier work and present new TP probes for intracellular free Zn ions (AZn1 and AZn2) derived from 2-acetyl-6-(dimethylamino)naphthalene (acedan) as the fluorophore and N,N-di-(2-picolyl)ethylenediamine (DPEN) as the Zn chelator. Acedan is a polarity-sensitive fluorophore that has been successfully employed in the design of TP fluorescent probes for the membrane and metal ions, and DPEN is a well-known receptor for Zn. Herein, we report that AZn1 and AZn2 are capable of imaging the intracellular free Zn ions in live cells for a long period of time and in living tissue at a depth of > 80 mm without mistargeting and photobleaching problems. The synthesis of AZn1 and AZn2 is shown in Scheme 1. The water solubilities of AZn1 and AZn2 are about 3.0 mm, which is sufficient for staining the cell (see the Supporting

Detection of mercury in fish organs with a two-photon fluorescent probe

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.

A Two-Photon Tracer for Glucose Uptake†

Glucose is the principal energy source essential for cell growth. Fast-growing cancer cells exhibit a high rate of glycolysis; hence, the rate of glucose uptake is faster in these cells, primarily due to overexpression or enhanced intracellular translocation of glucose transporters (GLUTs) and increased activity of mitochondria-bound hexokinases in the tumor. To monitor glucose metabolism in living systems, a variety of tracers, such as [F]-2-fluoro-2-deoxyglucose (FDG), 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2deoxy-d-glucose (2-NBDG; Scheme 1), and IR dye 800CW-2DG, have been developed. FDG is widely used in the in vivo analysis of glucose metabolism by positron emission tomography (PET), whereas 2-NBDG and IR dye 800CW-2DG are fluorescent probes that have been used for studying cellular metabolic functions involving GLUTs and in tumorimaging studies. Recently, we developed a new fluorescent probe, cyanine-3-linked O-1-glycosylated glucose (Cy3-Glc-a ; Scheme 1), which is a better glucose probe than 2-NBDG because it can be used without glucose starvation, produces a much brighter image, and can be applied for the screening of anticancer agents. In one-photon microscopy (OPM), the probes are excited with short-wavelength light ( 350–550 nm); this, however, limits their application in tissue imaging, owing to inherent problems such as shallow penetration depth (< 80 mm), interference by cellular autofluorescence, photobleaching, and photodamage. 11] To overcome these problems, it is crucial to use two-photon microscopy (TPM), which utilizes two near-infrared photons for excitation. TPM offers a number of advantages over OPM, including greater penetration depth (> 500 mm), localized excitation, and longer observation times. In particular, the extra penetration depth afforded by TPM is an essential element for application in tissue-imaging studies because the artifacts arising from surface preparation, such as damaged cells, can extend over 70 mm into the tissue interior. However, visualization of glucose uptake by live cells and tissues with two-photon (TP) tracers has not been reported so far. The requirements for a TP tracer to visualize glucose uptake include sufficient water solubility for staining cells and tissues, preferential uptake by cancer cells, a large TP crosssection for a bright TPM image, pH resistance, and high photostability. Our strategy was to link a-d-glucose with the fluorophore 2-acetyl-6-dimethylaminonaphthalene (acedan) through 3,6-dioxaoctane-1,8-diamine or a piperazine linkage (in AG1 and AG2, respectively; Scheme 1), so that the tracers are transported into the cells through the glucose-specific mechanism. Acedan is a polarity-sensitive fluorophore that has been successfully employed in the development of TP probes for the cell membrane, metal ions, and acidic vesicles. We now report that these tracers facilitate the visualization of glucose uptake in cancer cells and live tissues at a depth of 75–150 mm for more than 3000 s and can be used for screening anticancer agents. The preparation of AG1 and AG2 is shown in Scheme 2. 6-Acetyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene Scheme 1. Structures of fluorescent tracers AG1, AG2, 2-NBDG, and Cy3-Gly-a.

A two-photon fluorescent probe for near-membrane calcium ions in live cells and tissues

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.

Two-photon probes for Zn2+ ions with various dissociation constants. Detection of Zn2+ ions in live cells and tissues by two-photon microscopy.

A series of 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.

A Two‐Photon Turn‐On Probe for Lipid Rafts with Minimum Internalization

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.

A two-photon turn-on probe for glucose uptake

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.