Fluorescence microscopy

Our group have been working for 20 years on developing and using single-molecule fluorescence methods based on observation of either diffusing or surface-immobilised molecules. Such methods offer a unique advantage over traditional ensemble techniques as they unveil the presence of any heterogeneity within a population, allow the observation of rare events, and reveal the dynamic nature of biological systems with high temporal and spatial resolution. We have been in particular using the methods of single-molecule FRET, single-molecule tracking, and localisation-based super-resolutions imaging.

Single-molecule FRET 

• Single-molecule Förster (or Fluorescence) Resonance Energy Transfer, or FRET) is a powerful biophysical technique that allows study of the structural dynamics and interactions of individual biomolecules at the nanoscale; it essentially acts as a molecular ruler for the 2-10 nm length scale.
• FRET typically relies on the transfer of energy between two fluorophores, one serving as a donor and the other as an acceptor, when they are in close proximity. In single-molecule FRET, a pair of fluorophores is attached to specific sites on a biomolecule of interest, such as a protein or nucleic acid. By monitoring the fluctuations in fluorescence intensity (or lifetime) of these individual fluorophores in real-time, we directly monitor conformational changes, molecular interactions, and structural dynamics with unprecedented spatial and temporal resolution.
• Single-molecule FRET has proven instrumental in advancing our understanding of complex biological processes, offering a unique perspective that reveal functionally important molecular heterogeneity and allows the observation of rare events that might be masked in ensemble measurements. Our lab has developed several smFRET methods (see below) and applied them to answer long-standing questions in many research areas, such as in bacterial gene expression and DNA repair, as well as in viral replication.

Single-particle tracking 

• Single-molecule tracking is a cutting-edge experimental biophysical technique that captures the motion of individual biomolecules in real time. This method involves labeling molecules of interest, such as proteins or nanoparticles, with fluorescent markers and then recording microscopy movies to precisely monitor the molecular movements at the single-molecule level.
• By capturing the trajectories and diffusion patterns of individual molecules, we can gain valuable insights into cellular processes, such as molecular transport, protein-protein interactions, and dynamic changes in cellular structures. This technique has applications across various fields, including cell biology, neuroscience, and drug discovery, contributing significantly to our understanding of the fundamental principles governing molecular behaviour within living organisms.
• We have developed novel methods to perform single-molecule tracking in solution as well as in live bacteria, and employed these methods to study the interaction of a wide-variety of DNA-binding machinery with the bacterial chromosome.

Single-molecule localisation microscopy (SMLM)

• SMLM is a revolutionary suite of microscopy techniques that use clever tricks to bypass the diffraction limit and enable researchers to visualize cellular structures and biomolecules with unprecedented resolution. Prominent methods in this category include stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT).

• In these techniques, fluorophores are activated and localized sequentially, allowing for the precise determination of their positions. By collecting thousands of these localized points, a “super-resolved” image is reconstructed, revealing intricate details beyond the diffraction limit. This method (which was awarded a Nobel Prize in Chemistry in 2014) has transformed our understanding of cellular architecture, providing insights into subcellular structures, organelles, and molecular interactions at nanoscale resolution.

• We have been using single-molecule imaging to study the spatial organisation of gene expression and DNA repair machinery in bacteria, as well as getting the nanoscale organisation of different types of the influenza virus.