Carbocyanine Dye Research Articles

Trimethyltin intoxication induces the migration of ventricular/subventricular zone cells to the injured murine hippocampus

Following the postnatal decline of cell proliferation in the mammalian central nervous system, the adult brain retains progenitor cells with stem cell-like properties in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus. Brain injury can stimulate proliferation and redirect the migration pattern of SVZ precursor cells to the injury site. Sublethal exposure to the neurotoxicant trimethyltin (TMT) causes dose-dependent necrosis and apoptosis in the hippocampus dentate gyrus and increases SGZ stem cell proliferation to generate new granule cells. To determine whether SVZ cells also contribute to the repopulation of the TMT-damaged dentate gyrus, 6-8 week old male C3H mice were injected with the carbocyanine dye spDiI and bromodeoxyuridine (80mg/kg; ip.) to label ventricular cells prior to TMT exposure. The presence of labeled cells in hippocampus was determined 7 and 28days after TMT exposure. No significant change in the number of BrdU+ and spDiI+ cells was observed in the dentate gyrus 7days after TMT treatment. However, 28days after TMT treatment there was a 3–4 fold increase in the number of spDiI-labeled cells in the hippocampal hilus and dentate gyrus. Few spDiI+ cells stained positive for the mature phenotypic markers NeuN or GFAP, suggesting they may represent undifferentiated cells. A small percentage of migrating cells were BrdU+/spDiI+, indicating some newly produced, SVZ- derived precursors migrated to the hippocampus. Taken together, these data suggest that TMT-induced injury of the hippocampus can stimulate the migration of ventricular zone-derived cells to injured dentate gyrus.

Lipoplexes for Gene Delivery Characterized by Fluorescence Correlation Spectroscopy

Liposomes composed of cationic lipids have been extensively investigated as drug delivery systems [1] and carrier for gene therapy and regenerative medicine as an alternative to viral approaches [2]. One exemplary regenerative medicine concept is the use of induced pluripotent stem cells (iPS) reprogrammed through the introduction of pluripotency genes such as OCT4, Sox2, KLF4 and c-Myc [3]. In this work, we study the complexation of adequate liposome formulations with these four genes through Fluorescence Cross-Correlation Spectroscopy (FCCS). [4]Liposomes stained with the carbocyanine dye 1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD) and fluorescently labelled plasmids carrying the OCT4, Sox2, KLF4 and c-Myc genes are characterized via single photon FCCS studies with a confocal microscope (LSM780, Zeiss, Oberkochen, Germany). Using CX-rhodamine and fluorescein as fluorescent labelling for the genes, we create two complementary wavelength windows to the far red wavelength window necessary to observe DiD fluorescence. Thereby, we create the possibility to monitor liposome loading with more than one gene.[1] Allen, T. M. & Cullis, P. R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Deliv. Rev. 65, 36-48 (2013).[2] Niidome, T. & Huang, L. Gene Therapy Progress and Prospects: Nonviral vectors. Gene Ther. 9, 1647-1652 (2002).[3] Takahashi, K. & Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126, 663-676 (2006).[4] Merkle, D., Lees-Miller, S. P. & Cramb, D. T. Structure and Dynamics of Lipoplex Formation Examined Using Two-Photon Fluorescence Cross-Correlation Spectroscopy. Biochemistry 43, 7263-7272 (2004).

Mapping multidimensional electronic structure and ultrafast dynamics with single-element detection and compressive sensing.

Compressive sensing allows signals to be efficiently captured by exploiting their inherent sparsity. Here we implement sparse sampling to capture the electronic structure and ultrafast dynamics of molecular systems using phase-resolved 2D coherent spectroscopy. Until now, 2D spectroscopy has been hampered by its reliance on array detectors that operate in limited spectral regions. Combining spatial encoding of the nonlinear optical response and rapid signal modulation allows retrieval of state-resolved correlation maps in a photosynthetic protein and carbocyanine dye. We report complete Hadamard reconstruction of the signals and compression factors as high as 10, in good agreement with array-detected spectra. Single-point array reconstruction by spatial encoding (SPARSE) Spectroscopy reduces acquisition times by about an order of magnitude, with further speed improvements enabled by fast scanning of a digital micromirror device. We envision unprecedented applications for coherent spectroscopy using frequency combs and super-continua in diverse spectral regions.

Fluorimetric Methods for Analysis of Permeability, Drug Transport Kinetics, and Inhibition of the ABCB1 Membrane Transporter.

The cell membrane P-glycoprotein (P-gp; MDR1, ABCB1) is an energy-dependent efflux pump that belongs to the ATP-binding cassette (ABC) family of transporters, and has been associated with drug resistance in eukaryotic cells. Multidrug resistance (MDR) is related to an increased expression and function of the ABCB1 (P-gp) efflux pump that often causes chemotherapeutic failure in cancer. Modulators of this efflux pump, such as the calcium channel blocker verapamil (VP) and cyclosporine A (CypA), can reverse the MDR phenotype but in vivo studies have revealed disappointing results due to adverse side effects. Currently available methods are unable to visualize and assess in a real-time basis the effectiveness of ABCB1 inhibitors on the uptake and efflux of ABCB1 substrates. However, predicting and testing ABCB1 modulation activity using living cells during drug development are crucial. The use of ABCB1-transfected mouse T-lymphoma cell line to study the uptake/efflux of fluorescent probes like ethidium bromide (EB), rhodamine 123 (Rh-123), and carbocyanine dye DiOC2, in the presence and absence of potential inhibitors, is currently used in our laboratories to evaluate the ability of a drug to inhibit ABCB1-mediated drug accumulation and efflux. Here we describe and compare three in vitro methods, which evaluate the permeability, transport kinetics of fluorescent substrates, and inhibition of the ABCB1 efflux pump by drugs of chemical synthesis or extracted from natural sources, using model cancer cell lines overexpressing this transporter, namely (1) real-time fluorimetry that assesses the accumulation of ethidium bromide, (2) flow cytometry, and (3) fluorescent microscopy using rhodamine 123 and DiOC2.

Selective recognition of G-quadruplexes by a dimeric carbocyanine dye

A novel dimeric carbocyanine dye is found to recognise G-quadruplex structures selectively compared to mixed sequence or double-stranded DNA molecules.

Estimation of Candida albicans ABC Transporter Behavior in Real-Time via Fluorescence.

We present a fluorometric method for determining ABC transporter activity in the pathogenic fungus C. albicans during different growth phases and in response to glucose. The carbocyanine dye diS-C3(3) was previously used to monitor plasma membrane potentials and test the influence of surface-active compounds in membrane polarization. We used diS-C3(3) to show changes in fluorescence kinetics that reflect changes in the activity of ABC transporters in C. albicans growth. Cdr1-GFP fluorescence, revealed that Cdr1p relocates to the inside of the cell after the early-log growth phase. Addition of glucose to the cell suspension resulted in Cdr1p transporter expression in the CDR2-knockout strain. We confirmed the diS-C3(3) results by standard RT-PCR and Western blotting.

The carbocyanine dye DiD labels in vitro and in vivo neural stem cells of the subventricular zone as well as myelinated structures following in vivo injection in the lateral ventricle.

Carbocyanines are fluorescent lipophilic cationic dyes used since the early 1980s as neuronal tracers. Several applications of these compounds have been developed thanks to their low cell toxicity, lateral diffusion within the cellular membranes, and good photostability. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine 4-chlorobenzenesulfonate (DiD) is an interesting component of this family because, in addition to the classic carbocyanine properties, it has a longer wavelength compared with its analogues. That makes DiD an excellent carbocyanine for labeling cells and tissues with significant intrinsic fluorescence. Drug encapsulation, drug delivery, and cellular transplantation are also fields using DiD-based systems where having detailed knowledge about its behavior as a single entity is important. Recently, promising studies concerned neural stem cells from the subventricular zone of the lateral ventricle in the brain (their natural niche) and their potential therapeutic use. Here, we show that DiD is able to label these stem cells in vitro and present basilar information concerning its pharmacokinetics, concentrations, and microscope protocols. Moreover, when DiD is injected in vivo in the cerebrospinal fluid present in the lateral ventricle of rat, it also labels stem cells as well as myelinated structures of the caudoputamen. This analysis provides a database to consult when planning experiments concerning DiD and neural stem cells from the subventricular zone.

Inter-nanocarrier and nanocarrier-to-cell transfer assays demonstrate the risk of an immediate unloading of dye from labeled lipid nanocapsules

Release studies constitute a fundamental part of the nanovector characterization. However, it can be difficult to correctly assess the release of lipophilic compounds from lipid nanocarriers using conventional assays. Previously, we proposed a method including an extraction with oil to measure the loading stability of lipophilic dyes in lipid nanocapsules (LNCs). The method indicated a rapid release of Nile Red from LNCs, while the loading of lipophilic carbocyanine dyes remained stable. This method, although interesting for a rapid screening of the fluorescence labeling stability of nanocarriers, is far from what happens in vivo, where lipid acceptor phases are nanostructured. Here, lipophilic dye loading stability has been assessed, by monitoring dye transfer from LNCs toward stable colloidal lipid nanocompartments, i.e. non-loaded LNCs, using new methodology based on size exclusion chromatography (SEC) and Förster Resonance Energy Transfer (FRET). Dye transfer between LNCs and THP-1 cells (as model for circulating cells) has also been studied by FACS. The assays reveal an almost instantaneous transfer of Nile Red between LNCs, from LNCs to THP-1 cells, between THP-1 cells, and a reversal transfer from THP-1 cells to LNCs. On the contrary, there was no detectable transfer of the lipophilic carbocyanine dyes. Dye release was also analyzed using dialyses, which only revealed a very slow release of Nile Red from LNCs, demonstrating the weakness of membrane based assays for investigations of the lipophilic compound loading stability in lipid nanocarriers. These results highlight the importance of using relevant release assays, and the potential risk of an immediate unloading of lipophilic fluorescent dyes from lipid nanocarriers, in the presence of a lipid acceptor nanocompartment. Some misinterpretations of cellular trafficking and in vivo biodistribution of fluorescent nanoparticles should be avoided.

Near-infrared fluorescence heptamethine carbocyanine dyes mediate imaging and targeted drug delivery for human brain tumor

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood–brain and blood–tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Tumor‐Penetrating Nanotherapeutics Loading a Near‐Infrared Probe Inhibit Growth and Metastasis of Breast Cancer

The tumor growth and metastasis is the leading reason for the high mortality of breast cancer. Herein, it is first reported a deep tumor‐penetrating photothermal nanotherapeutics loading a near‐infrared (NIR) probe for potential photothermal therapy (PTT) of tumor growth and metastasis of breast cancer. The NIR probe of 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindotricarbocyanine iodide (DiR), a lipophilicfluorescent carbocyanine dye with strong light‐absorbing capability, is entrapped into the photothermal nanotherapeutics for PTT application. The DiR‐loaded photothermal nanotherapeutics (DPN) is homogeneous nanometer‐sized particles with the mean diameter of 24.5 ± 4.1 nm. Upon 808 nm laser irradiation, DPN presents superior production of thermal energy than free DiR both in vitro and in vivo. The cell proliferation and migration activities of metastatic 4T1 breast cancer cells are obviously inhibited by DPN in combination with NIR irradiation. Moreover, DPN can induce a higher accumulation in tumor and penetrate into the deep interior of tumor tissues. The in vivo PTT measurements indicate that the growth and metastasis of breast cancer are entirely inhibited by a single treatment of DPN with NIR irradiation. Therefore, the deep tumor‐penetrating DPN can provide a promising strategy for PTT of tumor progression and metastasis of breast cancer.

Multispectral labeling of embryonic cells with lipophilic carbocyanine dyes.

Incubation of hatched Strongylocentrotus purpuratus sea urchin embryos or larvae with suspensions of the carbocyanine dyes DiI, DiO, and DiD resulted in the random labeling of membranes of some ectodermal epithelial cells and blastocoelar cells, producing a range of differentially colored cells that can be tracked during development. Simultaneous application of soluble Vybrant® preparations of the three dyes resulted in similar labeling of each cell. Dye labeling of the ectoderm was nearly eliminated by deciliation and some ciliated squamous epithelial cells adjacent to labelled cells were refractory to Vybrant® dye uptake irrespective of concentration or duration of treatment, together suggesting local variation in the properties of cell membranes or cilia. Furthermore, single cells possessing distinctive morphological features were detected.

Fluorescence properties of indolenium carbocyanine dyes in solid state

The fluorescence intensity of indolenium carbocyanine dyes in the solid state depended on both the alkyl group on the nitrogen atoms in the indolenium rings and the kind of counter anions. The N-propyl derivative having a bis(trifluoromethylsulfonyl)imide anion, whose fluorescence maximum was observed at 657 nm with shoulder peaks, exhibited the highest fluorescence quantum yield of 0.31. The wave deconvolution of fluorescence spectrum indicated three components assigned to the S1,0→S0,0, S1,0→S0,1, and S1,0→S0,2 transitions. The X-ray structure of this compound indicates an isolated monomer-type packing, whereas the other derivatives shows the isolated dimer-type packing or consequent π/π stacking. The cyanine dye having the least π/π interactions among adjacent cationic fluorophores could exhibit the most intense fluorescence in the solid state.

Neural basis of the stress response in a pufferfish, Takifugu obscurus.

When faced with danger, pufferfish react with both a fast-start escape response and inflation behavior. The neural basis of these stress responses in the pufferfish has not been described. The present study reveals that during inflation behavior, the buccal cavity expands and compresses as a pump to control the direction of water flow and draws water in and out. The inflation involves a series of major anatomical modifications of the head. The greatly enlarged first branchiostegal ray and its associated hyohyoideus abductor muscle are the key mechanisms responsible for this behavior. The nerve branch innervating the hyohyoideus abductor muscle is highly developed, and its central origin at the motor nucleus of the VIIth cranial nerve was revealed by tract-tracing using the carbocyanine dye DiI. The central connections from its origin were found to be several motor nuclei in the medulla and the spinal cord, the nuclei of cranial nerve III and IV in the mesencephalon, and the pretectalis superficialis and periventricular preoptic nuclei in the diencephalon. Both the sympathetic cells and the parvocellular part of the periventricular preoptic nucleus might play a neuro-endocrine role in the rapid movement of the buccal cavity during puffing behavior in this species of pufferfish. The central circuit revealed by this study is hypothesized to mediate the C-start escape behavior and puffing behavior.

Electronic excitation energy transfer between molecules of cyanine dyes in a complex with protein and in systems of magnetic nanoparticles with protein coatings

Electronic excitation energy transfer (EEET) between molecules of carbocyanine dyes bound noncovalently with human serum albumin (HSA) has been studied. 3,3′-Di-(gamma-sulfopropyl)-9-methylcarbocyanine betaine has been used as an electronic excitation energy donor and 3,3′-di-(gamma-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethylthiacarbocyanine betaine, as an acceptor dye. EEET has been studied both in samples of HSA solutions and in systems containing protein-coated magnetic nanoparticles. Stable coatings of magnetic nanoparticles, consisting of a mixture of HSA and bovine serum albumin, have been formed by the method of free radical linking of protein molecules adsorbed on the surface of the particles. The effect of HSA denaturation on the spectral and fluorescent properties of the dyes and EEET has been studied, and the data on donor fluorescence quenching by the acceptor have been obtained. It has been concluded that HSA retains its functional properties in the linked coating, thereby providing evidence for biocompatibility of the coatings obtained by free-radical oxidative protein modification.

Primary Acute Lymphoblastic Leukemia Cells Create a Drug-Resistant Niche By Tunneling Nanotube Signaling with Mesenchymal Stromal Cells

Background: Acute lymphoblastic leukemia (ALL) cells reside in the bone marrow microenvironment, which nurtures and protects cells from chemotherapeutic drugs. Bidirectional signaling between leukemic cells and their niche is shown to be essential for these processes. A major gap in our knowledge is the lack of insight into the functional mechanism regulating and controlling this crosstalk. Tunneling nanotubes (TNTs) have recently been reported as a novel mode of communication between eukaryotic cells, which can facilitate the transport of several types of cellular cargo.Objectives: This study aims to investigate the presence and functional importance of TNTs in the leukemic niche.Results: Here, we show for the first time that TNT signaling occurs between primary patient-derived leukemic cells and primary mesenchymal stromal cells using flow cytometry and time-lapse confocal microscopy. TNTs form within minutes after the start of co-culture and efficiently transfer lipophilic carbocyanine dye DiI from ALL cells towards MSCs. Dye transfer was significantly reduced when TNT signaling was inhibited using three independent experimental setups: actin inhibition, mechanical disruption through gentle shaking of cell cultures, and physical separation using a 3.0 µm pore-sized transwell system (4-fold, 5-fold and 35-fold reduction, respectively). In reciprocal experiments we also observed dye transfer via TNTs from MSCs to BCP-ALL cells, but the magnitude was strikingly less (175-fold, p ≤ 0.001). In addition, we show that leukemic cells use TNTs to modulate their microenvironment by directing non-malignant stromal cells to produce pro-survival cytokines. We observed patient-specific cytokine signatures after co-culture of primary ALL cells with different primary MSCs. For example, IP10/CXCL10 levels increased more than 1000-fold when patient ALL#1 cells were co-cultured with primary MSCs, but were undetectable in co-cultures from patient ALL#2 cells. Induction of all cytokines was dependent on TNT signaling, as TNT inhibition significantly lowered cytokine production (p ≤ 0.001). Importantly, we show that TNT signaling is functionally important for leukemic cell survival and stroma-driven drug resistance. Leukemic cell viability was assessed by flow cytometry after staining with Annexin V, Propidium Iodide and CD19. Primary leukemic cell survival significantly increased in 5-day co-cultures with primary MSCs compared to mono-culture (p ≤ 0.001). When TNT formation was prevented by shaking of these co-cultures or by transwell conditions, the cell viability reduced 3.5- and 3.6-fold, respectively (p ≤ 0.001). TNT inhibition similarly reduced the survival of primary ALL cells in co-culture with MSCs during prednisolone exposure (p ≤ 0.01). Co-culture with primary MSCs also induced resistance to prednisolone 2.5-fold compared to mono-culture in a proliferative setting, using the BCP-ALL cell line NALM6. Inhibition of TNT formation by shaking or transwell conditions significantly reduced this effect: cells in co-culture were only 1.4- and 1.1-fold more resistant compared to mono-culture, respectively (p ≤ 0.01).Conclusion: The presented study identifies TNT formation as a major regulator of interaction between ALL cells and their bone marrow niche, which facilitates signaling mainly from leukemic cells towards MSCs. This signaling drives the release of cytokines within the microenvironment. Disruption of tunneling nanotubes inhibits this release, diminishes the survival benefit that MSCs provide to primary ALL cells, and sensitizes ALL cells to the important anti-leukemic drug prednisolone. This observation gives insight into the pathogenesis of BCP-ALL and opens new avenues to develop more effective therapies that interfere with the leukemic niche. DisclosuresNo relevant conflicts of interest to declare.

Atomic force and scanning near-field optical microscopy study of carbocyanine dye J-aggregates

Atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) imaging of fibrillar J-aggregates of carbocyanine dye 3,3′-bis(γ-sulfopropyl)-5,5′,6,6′-tetrachloro-1,1′-dioctylbenzimidacarbocyanine (C8S3) is performed on mica and glass substrates at room temperature. High resolution AFM imaging reveal that thick fibrils consist of narrow sub-fibrils. A model of an elementary fibril with a cross section 3 × 4 nm2 in size is proposed. The SNOM measurements made in the contact mode indicate the high mechanical strength of the fibrillar J-aggregates.

Near‑infrared fluorescence imaging of prostate cancer using heptamethine carbocyanine dyes.

Near-infrared fluorescence (NIRF) imaging is an attractive novel modality for the detection of cancer. A previous study defined two organic polymethine cyanine dyes as ideal NIRF probes, IR-783 and its derivative MHI-148, which have excellent optical characteristics, superior biocompatibility and cancer targeting abilities. To investigate the feasibility of NIRF dye-mediated prostate cancer imaging, dye uptake and subcellular co-localization were investigated in PC-3, DU-145 and LNCaP human prostate cancer cells and RWPE-1 normal prostate epithelial cells. Different organic anion transporting peptide (OATP) inhibitors were utilized to explore the potential role of the OATP subtype, including the nonspecific OATP inhibitor bromosulfophthalein, the OATP1 inhibitor 17β-estradiol, the selective OATP1B1 inhibitor rifampicin and the selective OATP1B3 inhibitor cholecystokinin octapeptide. NIRF dyes were also used for the simulated detection of circulating tumor cells and the rapid detection of prostate cancer in human prostate cancer tissues and prostate cancer xenografts in mouse models. The results revealed that the cancer-specific uptake of these organic dyes in prostate cancer cells occurred primarily via OATP1B3. A strong NIRF signal was detected in prostate cancer tissues, but not in normal tissues that were stained with IR-783. Prostate cancer cells were recognized with particular NIR fluorescence in isolated mononuclear cell mixtures. The results of the present study demonstrated that NIRF dye-mediated imaging is a feasible and practicable method for prostate cancer detection, although further investigative studies are required before clinical translation.

In situ synthesis of semiconductor nanocrystals at the surface of tubular J-aggregates

Hybrid nanostructures consisting of dye molecules and semiconducting nanocrystals are currently much investigated because they can benefit from the strong absorption of organic dyes and the chemical stability of inorganic components, a combination of properties that is promising for various optoelectronic applications. Here, we report an in situ synthesis method to design hybrid nanotubes by growing metal sulfide (MeS) nanocrystals at the surface of tubular J-aggregates of amphiphilic carbocyanine dyes. In this method, metal cations (Me2+ = Cd2+, Zn2+, and Ni2+) are enriched at the negatively charged surface of J-aggregates via electrostatic attraction, giving rise to a diffuse Helmholtz layer. Subsequent addition of thioacetamide and ammonium hydroxide leads to the formation of a dense layer of MeS nanocrystals exclusively close to or at the surface of J-aggregates. Typically, a shell of CdS nanocrystals (size ∼ 3 nm) is formed at the surface of tubular J-aggregates without significantly affecting the aggregate morphology. The crystal structure is analyzed by electron diffraction revealing a sphalerite structure. The coverage of CdS nanocrystals on J-aggregates can be tuned by varying the concentration of the sulphur source. Efficient energy transfer from CdS nanocrystals to J-aggregates results in the quenching of the CdS photoluminescence. Structurally and morphologically similar hybrid systems of tubular J-aggregates and semiconductor nanocrystals (e.g., ZnS and NiS) are synthesized by analog chemical reactions.

Direct Inhibition of Choline Kinase by a Near-Infrared Fluorescent Carbocyanine

Choline kinase alpha (ChoK) expression is increasingly being recognized as an important indicator of breast cancer prognosis; however, previous efforts to noninvasively measure ChoK status have been complicated by the spectral limitations of in vivo magnetic resonance spectroscopy (MRS) and the complex network of enzymes involved in choline metabolism. The most effective ChoK inhibitors are symmetric and contain quaternary ammonium groups within heterocyclic head groups connected by an aliphatic spacer. Characterization of these bis-pyridinium and bis-quinolinium compounds has led to phase I clinical trials to assess small-molecule inhibitors of ChoK for solid tumor treatment. We report the development of a novel carbocyanine dye, JAS239, whose bis-indolium structure conforms to the parameters established for ChoK specificity and whose spacer length confers fluorescence in the near-infrared (NIR) window. Fluorimetry and confocal microscopy were used to demonstrate that JAS239 rapidly enters breast cancer cells independent of the choline transporters, with accumulation in the cytosolic space where ChoK is active. Radio-tracing and (1)H MRS techniques were used to determine that JAS239 binds and competitively inhibits ChoK intracellularly, preventing choline phosphorylation while inducing cell death in breast cancer cell lines with similar efficacy to known ChoK inhibitors. Fluorescent molecules that report on ChoK status have potential use as companion diagnostics for noninvasive breast tumor staging, because NIR fluorescence allows for detection of real-time probe accumulation in vivo. Furthermore, their ability as novel ChoK inhibitors may prove effective against aggressive, therapy-resistant tumors.

Atomic Force and Scanning Near-Field Optical Microscopy Study of Carbocyanine Dye J-aggregates

Atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) imaging of fibrillar J-aggregates of carbocyanine dye 3,3′-bis(γ-sulfopropyl)-5,5′,6,6′-tetrachloro-1,1′-dioctylbenzimidacarbocyanine (C8S3) is performed on mica and glass substrates at room temperature. High resolution AFM imaging reveal that thick fibrils consist of narrow sub-fibrils. A model of an elementary fibril with a cross section 3 × 4 nm2 in size is proposed. The SNOM measurements made in the contact mode indicate the high mechanical strength of the fibrillar J-aggregates.