FRETANDFORCE · Single molecule studies of DNA Polymerases using Fluorescence Microscopy and laser tweezers. A step towards the combination of these techniques

Final Activity and Management Report Summary - FRETANDFORCE (Single Molecule Studies of DNA Polymerases Using Fluorescence Microscopy and Laser Tweezers ...)

Although single molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K), the field has since advanced to include room temperature observations largely due to the use of high numerical aperture objective lenses, brighter fluorophores, and more sensitive detectors. This has opened the door for many chemical and biological systems to be studied at native temperatures at the single molecule level both in vitro and in vivo. However, systems and phenomena that operate at temperatures above 37 C remain difficult to study at the single molecule level due to the need for index matching fluids with high numerical aperture (NA) objective lenses. These fluids act as a thermal conductor between the sample and the objective and sustained exposure to high temperature can cause the objective to fail. This has prevented the single molecule study of thermophilic organisms, the interactions of their protein repertoire, and the temperature-dependent unfolding kinetics of nucleic acids and proteins. Here we report that high index of refraction micron-sized colloidal lenses are capable of achieving single molecule imaging at 70 C by incorporating a focusing element in immediate proximity to an emitting molecule; the optical system is completed by a low numerical aperture optic which can have a long working distance and an air interface. TiO2 colloidal lenses were used for parallel imaging of surface-immobilised single fluorophores and to measure real-time single molecule mesophilic and thermophilic DNA polymerase strand displacement replication through an immobilised template at 23 C and 70 C, respectively. Microfluidics is a rapidly emerging field which aims to create lab-on-chip capability for bioanalysis and other applications. Several researchers have considered integrating optics in the microfluidic environment, which has spawned the field of optofluidics. An important capability of optics is provided by the optical scattering force, which allows for particle manipulation and optical trapping. While light microscopy is suited to monitor changes in cell shape in a 2D representation, Scanning Ion Conductance microscopy provides quantitative 3D-information at a cellular or even subcellular level. Such studies are normally performed on multiple cells grown or fixed on a surface which leaves a question about the influence of surface adhesion and cell to cell heterogeneities and interactions upon the biochemical state of the cells studied. In order to create an optical trap which is independent of the imaged sample region we used a principle in which two counter propagating divergent laser beams create an optical trap in which cells can be trapped, rotated and imaged without touching or modifying them. Using this approach we realise a microfluidic flow system where cells can be trapped through a dual beam trapping region where a controlled rotation tales place and simultaneously recording 3D topographic images of living cells in real time is achieved using a scanning ion conductance microscope.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз