Development of fan‐shaped tracker for single particle tracking

With the development of super‐resolution fluorescence microscopy, complex dynamic processes in living cells can be observed and recorded with unprecedented temporal and spatial resolution. Single particle tracking (SPT) is the most important step to explore the relationship between the spatio‐temporal dynamics of subcellular molecules and their functions. Although previous studies have developed SPT algorithms to quantitatively analyze particle dynamics in cell, traditional tracking methods have poor performance when dealing with intersecting trajectories. This can be attributed to two main reasons: (a) they do not have point compensation process for overlapping objects; (b) they use inefficient motion prediction models. In this paper, we present a novel fan‐shaped tracker (FsT) algorithm to reconstruct the trajectories of subcellular vesicles in living cells. We proposed a customized point compensation method for overlapping objects based on the fan‐shaped motion trend of the particles. Furthermore, we validated the performance of the FsT in both simulated time‐lapse movies with variable imaging quality and in real vesicle moving images. Meanwhile, we compared the performance of FsT with other five state‐of‐the‐art tracking algorithms by using commonly defined measures. The results showed that our FsT achieves better performance in high signal‐to‐noise ratio conditions and in tracking of overlapping objects. We anticipate that our FsT method will have vast applications in tracking of moving objects in cell.     

Optimization of Pathogen Capture in Flowing Fluids with Magnetic Nanoparticles

Magnetic nanoparticles have been employed to capture pathogens for many biological applications; however, optimal particle sizes have been determined empirically in specific capturing protocols. Here, a theoretical model that simulates capture of bacteria is described and used to calculate bacterial collision frequencies and magnetophoretic properties for a range of particle sizes. The model predicts that particles with a diameter of 460 nm should produce optimal separation of bacteria in buffer flowing at 1 L h⁻¹. Validating the predictive power of the model, Staphylococcus aureus is separated from buffer and blood flowing through magnetic capture devices using six different sizes of magnetic particles. Experimental magnetic separation in buffer conditions confirms that particles with a diameter closest to the predicted optimal particle size provide the most effective capture. Modeling the capturing process in plasma and blood by introducing empirical constants (c_e), which integrate the interfering effects of biological components on the binding kinetics of magnetic beads to bacteria, smaller beads with 50 nm diameters are predicted that exhibit maximum magnetic separation of bacteria from blood and experimentally validated this trend. The predictive power of the model suggests its utility for the future design of magnetic separation for diagnostic and therapeutic applications.     

Engineering a Bacterial Tape Recorder

A method has been developed to produce and integrate single‐stranded DNA into genomic locations in bacteria in response to exogenous signals. The system functions similarly to a cellular tape recorder by writing information into DNA and reading it at a later time. Much like other cellular memory platforms, its operation is based on DNA recombinase function. However, the scalability and recording capacity have been improved over previous designs. In addition, memory storage was reversible and could be recorded in response to analogue inputs, such as light exposure. This modular memory writing system is an important addition to the genomic editing toolbox available for synthetic biology.     

Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb³⁺ and from sensitized Tb³⁺ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.