Preassembled Fluorescent Multivalent Probes for the Imaging of Anionic Membranes.

A new self-assembly process known as Synthavidin (synthetic avidin) technology was used to prepare targeted probes for near-infrared fluorescence imaging of anionic membranes and cell surfaces, a hallmark of many different types of disease. The probes were preassembled by threading a tetralactam macrocycle with six appended zinc-dipicolylamine (ZnDPA) targeting units onto a linear scaffold with one or two squaraine docking stations to produce hexavalent or dodecavalent fluorescent probes. A series of liposome titration experiments showed that multivalency promoted stronger membrane binding by the dodecavalent probe. In addition, the dodecavalent probe exhibited turn-on fluorescence due to probe unfolding during fluorescence microscopy at the membrane surface. However, the dodecavalent probe also had a higher tendency to self-aggregate after membrane binding, leading to probe self-quenching under certain conditions. This self-quenching effect was apparent during fluorescence microscopy experiments that recorded low fluorescence intensity from anionic dead and dying mammalian cells that were saturated with the dodecavalent probe. Conversely, probe self-quenching was not a factor with anionic microbial surfaces, where there was intense fluorescence staining by the dodecavalent probe. A successful set of rat tumor imaging experiments confirmed that the preassembled probes have sufficient mechanical stability for effective in vivo imaging. The results demonstrate the feasibility of this general class of preassembled fluorescent probes for multivalent targeting, but fluorescence imaging performance depends on the specific physical attributes of the biomarker target, such as the spatial distance between different copies of the biomarker and the propensity of the probe-biomarker complex to self-aggregate.

Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.

A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with 12 bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 h in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation.

Rapid Macrocycle Threading by a Fluorescent Dye-Polymer Conjugate in Water with Nanomolar Affinity.

A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethylene glycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with k(on) ≈ 10(6)-10(7) M(-1) s(-1). The association is effective under cell culture conditions and produces large changes in dye optical properties including turn-on near-infrared fluorescence that can be imaged using cell microscopy. Association constants in water are ∼1000 times higher than those in organic solvents and strongly enthalpically favored at 27 °C. The threading rate is hardly affected by the length of the PEG chains that flank the squaraine dye. For example, macrocycle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethylene glycol chains that are 20 times shorter. The results are a promising advance toward synthetic mimics of streptavidin/biotin.

HindSight: Encouraging Exploration through Direct Encoding of Personal Interaction History.

Physical and digital objects often leave markers of our use. Website links turn purple after we visit them, for example, showing us information we have yet to explore. These "footprints" of interaction offer substantial benefits in information saturated environments - they enable us to easily revisit old information, systematically explore new information, and quickly resume tasks after interruption. While applying these design principles have been successful in HCI contexts, direct encodings of personal interaction history have received scarce attention in data visualization. One reason is that there is little guidance for integrating history into visualizations where many visual channels are already occupied by data. More importantly, there is not firm evidence that making users aware of their interaction history results in benefits with regards to exploration or insights. Following these observations, we propose HindSight - an umbrella term for the design space of representing interaction history directly in existing data visualizations. In this paper, we examine the value of HindSight principles by augmenting existing visualizations with visual indicators of user interaction history (e.g. How the Recession Shaped the Economy in 255 Charts, NYTimes). In controlled experiments of over 400 participants, we found that HindSight designs generally encouraged people to visit more data and recall different insights after interaction. The results of our experiments suggest that simple additions to visualizations can make users aware of their interaction history, and that these additions significantly impact users' exploration and insights.

Selective photothermal inactivation of cells labeled with near-infrared croconaine dye.

Photothermal inactivation of cells caused by laser heating of a near-infrared croconaine dye is more effective when the dye is located inside the cell. The cell inactivation is spatially confined - laser irradiation of a mixed population of two different cell lines produces selective inactivation of the cells labeled with croconaine dye and does not harm adjacent unlabeled cells.

High Affinity Macrocycle Threading by a Near-Infrared Croconaine Dye with Flanking Polymer Chains.

Croconaine dyes have narrow and intense absorption bands at ∼800 nm, very weak fluorescence, and high photostabilities, which combine to make them very attractive chromophores for absorption-based imaging or laser heating technologies. The physical supramolecular properties of croconaine dyes have rarely been investigated, especially in water. This study focuses on a molecular threading process that encapsulates a croconaine dye inside a tetralactam macrocycle in organic or aqueous solvent. Macrocycle association and rate constant data are reported for a series of croconaine structures with different substituents attached to the ends of the dye. The association constants were highest in water (Ka ∼ 10(9) M(-1)), and the threading rate constants (kon) increased in the solvent order H2O > MeOH > CHCl3. Systematic variation of croconaine substituents located just outside the croconaine/macrocycle complexation interface hardly changed Ka but had a strong influence on kon. A croconaine dye with N-propyl groups at each end of the structure exhibited a desirable mixture of macrocycle threading properties; that is, there was rapid and quantitative croconaine/macrocycle complexation at relatively high concentrations in water, and no dissociation of the preassembled complex when it was diluted into a solution of fetal bovine serum, even after laser-induced photothermal heating of the solution. The combination of favorable near-infrared absorption properties and tunable mechanical stability makes threaded croconaine/macrocycle complexes very attractive as molecular probes or as supramolecular composites for various applications in absorption-based imaging or photothermal therapy.

Improving Bayesian Reasoning: The Effects of Phrasing, Visualization, and Spatial Ability.

Decades of research have repeatedly shown that people perform poorly at estimating and understanding conditional probabilities that are inherent in Bayesian reasoning problems. Yet in the medical domain, both physicians and patients make daily, life-critical judgments based on conditional probability. Although there have been a number of attempts to develop more effective ways to facilitate Bayesian reasoning, reports of these findings tend to be inconsistent and sometimes even contradictory. For instance, the reported accuracies for individuals being able to correctly estimate conditional probability range from 6% to 62%. In this work, we show that problem representation can significantly affect accuracies. By controlling the amount of information presented to the user, we demonstrate how text and visualization designs can increase overall accuracies to as high as 77%. Additionally, we found that for users with high spatial ability, our designs can further improve their accuracies to as high as 100%. By and large, our findings provide explanations for the inconsistent reports on accuracy in Bayesian reasoning tasks and show a significant improvement over existing methods. We believe that these findings can have immediate impact on risk communication in health-related fields.

Sensitive Structural Control of Macrocycle Threading by a Fluorescent Squaraine Dye Flanked by Polymer Chains.

A macrocyclic tetralactam is threaded by a complementary squaraine dye that is flanked by two polyethylene glycol chains to produce a pseudorotaxane complex with favorable near-infrared fluorescence properties. The association thermodynamics and kinetics were measured for a homologous series of squaraines with different N-alkyl substituents at both ends of the dye. The results show that subtle changes in substituent steric size have profound effects on threading kinetics without greatly altering the very high association constant.

Thiosquaramides: pH switchable anion transporters.

The transport of anions across cellular membranes is an important biological function governed by specialised proteins. In recent years, many small molecules have emerged that mimick the anion transport behaviour of these proteins, but only a few of these synthetic molecules also display the gating/switching behaviour seen in biological systems. A small series of thiosquaramides was synthesised and their pH-dependent chloride binding and anion transport behaviour was investigated using 1H NMR titrations, single crystal X-ray diffraction and a variety of vesicle-based techniques. Spectrophotometric titrations and DFT calculations revealed that the thiosquaramides are significantly more acidic than their oxosquaramide analogues, with pKa values between 4.0 and 9.0. This led to the observation that at pH 7.2 the anion transport ability of the thiosquaramides is fully switched OFF due to deprotonation of the receptor, but is completely switched ON at lower pH.

Thiosquaraine rotaxanes: synthesis, dynamic structure, and oxygen photosensitization.

Thiosquaraine dyes have sulfur atoms instead of oxygens on the central squaraine core, and they are powerful singlet oxygen photosensitizers. Stability studies show that they are susceptible to attack by nucleophiles. This problem was circumvented by preparing a mechanically interlocked thiosquaraine rotaxane. NMR studies of the rotaxane indicate an unusual dynamic molecular structure due to a nonsymmetrical coconformation. Upon irradiation with red light, the thiosquaraine rotaxane generates the same amount of singlet oxygen as the known photosensitizer methylene blue.