Single-molecule tracking technologies for quantifying the dynamics of gene regulation in cells, tissue and embryos

ABSTRACT For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation.

Controlled Fluorescent Labelling of Metal Oxide Nanoparticles for Artefact-free Live Cell Microscopy

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. They are best investigated using non-invasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labelling of nanoparticles with fluorescent dyes. When performed inadequately, unbound fluorophores and inadvertently altered chemical and physical properties of the nanoparticles can, however, result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we here describe a minimal combination of experimental methods to enable artefact-free fluorescent labelling of metal-oxide nanoparticles - the largest subpopulation of nanoparticles by industrial production and applications - and demonstrate its application in the case of TiO2 nanotubes. We 1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labelling, and 2) assess stable binding of the fluorescent dye to nanomaterial, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials.

Lung Marginated and Splenic Murine Resident Neutrophils Constitute Pioneers in Tissue-Defense During Systemic E. coli Challenge

The rapid response of neutrophils throughout the body to a systemic challenge is a critical first step in resolution of bacterial infection such as Escherichia coli (E. coli). Here we delineated the dynamics of this response, revealing novel insights into the molecular mechanisms using lung and spleen intravital microscopy and 3D ex vivo culture of living precision cut splenic slices in combination with fluorescent labelling of endogenous leukocytes. Within seconds after challenge, intravascular marginated neutrophils and lung endothelial cells (ECs) work cooperatively to capture pathogens. Neutrophils retained on lung ECs slow their velocity and aggregate in clusters that enlarge as circulating neutrophils carrying E. coli stop within the microvasculature. The absolute number of splenic neutrophils does not change following challenge; however, neutrophils increase their velocity, migrate to the marginal zone (MZ) and form clusters. Irrespective of their location all neutrophils capturing heat-inactivated E. coli take on an activated phenotype showing increasing surface CD11b. At a molecular level we show that neutralization of ICAM-1 results in splenic neutrophil redistribution to the MZ under homeostasis. Following challenge, splenic levels of CXCL12 and ICAM-1 are reduced allowing neutrophils to migrate to the MZ in a CD29-integrin dependent manner, where the enlargement of splenic neutrophil clusters is CXCR2-CXCL2 dependent. We show directly molecular mechanisms that allow tissue resident neutrophils to provide the first lines of antimicrobial defense by capturing circulating E. coli and forming clusters both in the microvessels of the lung and in the parenchyma of the spleen.

Synthesis of fluorescently-labelled poly(2-ethyl-2-oxazoline)-protected gold nanoparticles

Gold nanoparticles (GNPs) protected by poly(2-ethyl-2-oxazoline) (POZ) of different molecular weights (Mw = 5, 50, 200 and 500 kDa) were synthesised and characterised by dynamic light scattering, nanoparticle tracking analysis, zeta potential measurement and transmission electron microscopy. It was established that the use of POZ with 50 kDa resulted in formation of GNPs with low polydispersity while POZ with greater molecular weights led to formation of more polydisperse GNPs. Fluorescent labelling of these nanoparticles was achieved through their reaction with polyethyleneglycol dithiol (8-12 kDa) as a linker molecule with subsequent reaction with 6-(iodoacetamido) fluorescein. The fluorescent nature of obtained GNPs was confirmed by the appearance of the fluorescence peak at 510 nm that is typical for fluorescein molecules and glowing of the aqueous solution under the UV irradiaton. The fluorescently-labelled GNPs are promising tool in biomedical application to monitor the biological systems using fluorescent microscopy.

Silicon-rhodamine isothiocyanate for fluorescent labelling

An efficient high yielding synthesis for silicon-rhodamines (SiR) led to silicon-rhodamine isothiocyanate (SITC) for facile fluorescent labeling in high-resolution imaging.

Dansyl-NA3 conjugates for glycoprotein detection through fluorescent tagging and native gel electrophoresis

A new approach to the fluorescent labelling of glycoproteins was developed utilizing the aldehyde-reactive N-amino anthranilic acid (NA3) moiety linked to a dansyl fluorophore. The presented synthetic method is based...