No abstract

Single molecule tracking provides unprecedented insights into diffusional processes of systems in life and material sciences. Determination of molecule positions with high accuracy and correct connection of the determined positions to tracks is a challenging task with, so far, no universal solution for single fluorescing molecules tackling the challenge of low signal-to-noise ratios, frequent blinking and photo bleaching. Thus, the development of novel algorithms for automatic single molecule fluorescence tracking is essential to analyse the huge amount of diffusional data obtained with single molecule widefield fluorescence microscopy. Here, we present a novel tracking model using a top-down polyhedral approach which can be implemented effectively using standard linear programming solvers. The results of our tracking approach are compared to the ground truth of simulated data with different diffusion coefficients, signal-to-noise ratios and particle densities. We also determine the dependency of blinking on the analysed distribution of diffusion coefficients. To confirm the functionality of our tracking method, the results of automatic tracking and manual tracking by a human expert are compared and discussed.

Monitoring of the formation of stable fluorescent nanoparticles from controlled mixing of a THF solution of poly(fluorene ethynylene)-block-poly(ethylene glycol) in a microfluidic laminar flow crossjunction by spatially resolved fluorescence spectroscopy reveals the time scale of particle formation as well as incorporation of small molecule guests and the role of solvent mixing.

The three-dimensional orientation monitoring of anisotropic nanoparticles during dynamic processes is a fundamental issue. Herein we show that incorporation of a single fluorescent reporter molecule is a promising concept toward this goal. As a model system, shape anisotropic single lamella polyethylene (PE) nanocrystals bearing one single fluorescent reporter molecule were prepared via ring-opening metathesis polymerization (ROMP) of highly ring-strained trans-cyclooctene ( tr COE) using a mixture of a dye-functionalized ruthenium-based initiator (1; perylene diimide (PDI) substituted Hoveyda−Grubbs second generation Ru alkylidene) and an appropriate excess of the unlabeled analogue (2; Hoveyda−Grubbs second generation Ru alkylidene) in aqueous microemulsion as a key step and subsequent exhaustive hydrogenation (>99.9%) of the main-chain unsaturated polymer in the nanoparticles to yield nanocrystals of high molecular weight, strictly linear PE (M n = 8 × 10 5 g mol −1 ; M w /M n = 1.4). TEM and AFM show a particle thickness of ca. 12 nm with a lateral extension of typically 45 nm. Comparable initiation kinetics of both complexes 1 and 2, which is a key requirement for this approach, were revealed by fluorescence spectroscopy studies (ΔH ‡ = 57.4 kJ mol −1 , ΔS ‡ = −73.0 J mol −1 K −1 for 1 vs ΔH ‡ = 63.6 kJ mol −1 , ΔS ‡ = −80.8 J mol −1 K −1 for 2 for the initiation with n-butyl vinyl ether, respectively). The labeled nanocrystals were characterized by means of single molecule fluorescence spectroscopy. Orientational analysis via defocused wide-field fluorescence microscopy (DWFM) revealed a fixed orientation of the chromophores within the nanocrystals, with their long molecular axis predominantly oriented parallel to the polar axis of the nanoparticles.

Thermoresponsive materials exhibit an enormous potential for tissue engineering, separation systems, and drug delivery. We investigated the diffusion of laponite clay nanoparticles, which serve as physical cross-linkers to achieve improved material properties in poly(N-isopropylacrylamide) (PNIPAM)-clay composite hydrogels close to the gel point. The networks are formed through physical interactions between PNIPAM chains and clay nanoparticles after these two components are mixed. In contrast to previous studies, a covalent labeling strategy was chosen to minimize the amount of free dyes in solution. Single-particle tracking of the labeled clay nanoparticles showed that their diffusion is anomalous at all temperatures used in this study, reflecting the viscoelastic behavior as a cross-linker. Stepwise heating from 24 to 38 °C resulted in a slight increase of the diffusion coefficient and the anomality parameter α up to the volume phase transition temperature of ca. 31 °C, which was followed by a significant drop of both parameters, reflecting strongly hindered motion of the collapsed nanoparticle aggregates.