Kyoung J. Lee

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

Two-Photon Fluorescent Turn-On Probe for Lipid Rafts in Live Cell and Tissue

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.

Two-photon fluorescent probes for acidic vesicles in live cells and tissue.

enzymes and secretory proteins exhibiting a variety of functions. To determine their functions, a variety of membrane-permeable fluorescent pH and lysosomal probes have been developed, some of which are commercially available. However, use of these probes with one-photon microscopy (OPM) requires excitation with short-wavelength light (ca. 350–550 nm) that limits their application in deeptissue imaging, owing to the shallow penetration depth (less than 80 mm) as well as to photobleaching, photodamage, and cellular autofluorescence. To overcome these problems, it is crucial to use two-photon microscopy (TPM). TPM employs two near-infrared photons for excitation and offers a number of advantages over OPM, including increased penetration depth (greater than 500 mm), localized excitation, and prolonged observation time. The extra penetration depth that TPM affords is of particular interest in tissue imaging, because surface preparation artifacts such as damaged cells extend over 70 mm into the tissue interior. However, most of the OP fluorescent probes presently used for TPM have small TP action cross sections (Fd) that limit their usage. Although a TP pH probe with appreciable Fd (ca. 42 GM) has been reported, the utility of this probe in TPM imaging has not been verified. Therefore, there is a need to develop an efficient TP probe that can visualize acidic vesicles deep inside tissue for a long period of time. To design an efficient TP probe for acidic vesicles, we chose acedan as the TP fluorophore, because acedan-derived TP probes for Mg (AMg1) and Ca (ACa1) exhibited high photostability as well as significant TP action cross sections for the bright TPM image at low probe concentration, thus allowing the detection of the metal ions deep inside live tissues for over 1100 s. We have introduced an aniline, o-methoxy aniline (pKa(BH ) 4), or tertiary amine (pKa(BH ) 10) substituent as the proton-binding site through an amide linkage to the fluorophore. It is expected that AH1 and AH2 would emit TP-excited fluorescence (TPEF) upon protonation at pH< 4, whereas AL1 would emit TPEF in the acidic vesicles, where it should accumulate as the protonated form. Herein, we report that these probes are capable of imaging the acidic vesicles in live cells and living tissues at greater than 100 mm depth without mistargeting and photobleaching problems. Moreover, AL1 can visualize the transportation of the acidic vesicles in the hippocampal cornu ammonis CA3 region for a long period of time with the use of TPM. AH1, AH2, and AL1 were prepared in 47–77% yields by reactions of 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene and a p-phenylenediamine derivative or N,Ndimethylethylenediamine (see the Supporting Information). The solubilities of AH1, AH2, and AL1 in water are in the range of 5.0–9.0 mm, which are sufficient to stain the cells (Figure S2 in the Supporting Information). The fluorescence spectra of AH1, AH2, and AL1 show gradual bathochromic shifts with solvent polarity (ET) in the order 1,4-dioxane< DMF<EtOH<H2O (Figure S1 and Table S1 in the Supporting Information). The large bathochromic shifts with increasing solvent polarity indicate the utility of these molecules as polarity probes. TP action cross section was determined by investigating the TPEFof the probes using fluorescein as the reference (see the Supporting Information). The TP action spectra of AH1, Scheme 1. The structures of AH1, AH2, and AL1.

Developing scheduling systems for Daewoo Shipbuilding: DAS project

Abstract Daewoo Shipbuilding Company, one of the largest shipbuilders in the world, had difficulties with planning and scheduling its production process. To solve the problems, Korea Advanced Institute of Science and Technology (KAIST) and Daewoo have been jointly performing the DAS (DAewoo Shipbuilding Scheduling) Project for three years, from 1991 to 1993. To develop the integrated scheduling systems, several technological breakthroughs were necessary such as hierarchical architecture between systems, constraint directed graph search, spatial scheduling, dynamic assembly line scheduling, and neural network based man-hours estimation. Besides these technological research issues, we adopted the phased development strategy, which consists of three phases of vision revelation, data dependent realization, and prospective enhancement. The DAS systems were successfully launched in January 1994 and are being actively used as indispensable systems in the shipyard resulting in a significant improvement in productivity and reengineering of the scheduling process.

A cross-national market segmentation of online game industry using SOM

Abstract To compete successfully in todays global online game markets, a cross-national analysis for market segmentation is becoming a more important issue, by which companies are able to understand their domestic and foreign loyal customers and concentrate their limited resources into the target customers. However, previous research methodologies for market segmentation were difficult to be conducted on a cross-national analysis because they were performed within a nation. Additionally, the traditional clustering methodologies have not provided a unique clustering nor determined the precise number of clusters. The purpose of our research is to develop a new methodology for cross-national market segmentation. We propose a two-phase approach (TPA) integrating statistical and data mining methods. The first phase is conducted by a statistical method (MCFA: multi-group confirmatory factor analysis) to test the difference between national clustering factors. The second phase is conducted by a data mining method (a two-level SOM) to develop the actual clusters within each nation. A two-level SOM is useful to effectively reduce the complexity of the reconstruction task and noise. Especially, our research tested the model with Korean and Japanese online game users because they are the frontier of global online game industries.

A spatial scheduling system and its application to shipbuilding: DAS-CURVE

Spatial scheduling considers not only traditional scheduling constraints like resource capacity and due dates, but also dynamic spatial layout of the objects. Automation of spatial scheduling is particularly important when the spatial resources are critical bottleneck resources, as is the case in the shipbuilding industry. To develop a spatial scheduling expert system for shipbuilding, a methodology for spatial layout of polygonal objects within rectangular plates is first developed. This study is then extended to the methodology for spatial scheduling, including the time dimension. The methodology is applied to the scheduling of Daewoo shipbuilding to build a system DAS-CURVE. DAS-CURVE is successfully operational and its experimental performance is remarkable.

Time-bound negotiation framework for electronic commerce agents

Abstract For efficient and informative coordination of agents especially in electronic commerce environment, a time-bound agent negotiation framework is proposed utilizing a time-based commitment scheme. By attaching commitment duration to agent messages, the traditional contract net protocol is extended to a time-bound negotiation framework (TBNF). The proposed negotiation framework has a new message type which allows for parties to agree upon the extension of a commitment duration, and a novel commitment concept in the form of negative commitment. The semantics of the messages with the commitment duration are interpreted, and then the three typical negotiation protocols are formally defined and compared — nothing-guaranteed protocol, acceptance-guaranteed protocol , and finite-time guarantee protocol — which can be incorporated into TBNF. The TBNF should provide a background for efficient and effective electronic commerce negotiation while accommodating each agents adaptive negotiation strategy.

Two-Photon Fluorescent Probes for Long-Term Imaging of Calcium Waves in Live Tissue

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.

Design of a RFID-Based ubiquitous comparison shopping system

With the spread of the so-called always-online environment that allows consumers to be online anytime, anyplace, the next step will be the integration of online and offline markets. Competition will be consequently further intensified and there is a probability that in the process the role of offline retailers will shift from that of a traditional retailer to a displayer role. When this occurs, appropriate technological devices and business models should be explored so that both displayers and retailers can benefit. To this end, this paper proposes an RFID (Radio Frequency Identification) technology-based pervasive comparison shopping business model. RFID will allow consumers to be seamlessly connected to the network, and the advent of a new shopping network will enable a smoothly functioning incentive mechanism between displayers and retailers. Ultimately, a new shopping network will enable consumers to be engaged in seamless commerce.

Superlattice, rhombus, square, and hexagonal standing waves in magnetically driven ferrofluid surface

Standing wave patterns that arise on the surface of ferrofluids by (single frequency) parametric forcing with an ac magnetic field are investigated experimentally. Depending on the frequency and amplitude of the forcing, the system exhibits various patterns including a superlattice and subharmonic rhombuses as well as conventional harmonic hexagons and subharmonic squares. The superlattice arises in a bicritical situation where harmonic and subharmonic modes collide. The rhombic pattern arises due to the nonmonotonic dispersion relation of a ferrofluid.