We have built a custom Total Internal Reflection Fluorescence (TIRF) microscope to enable the dynamic imaging of single molecules. Laser light is directed to the specimen through a microscope objective at a shallow angle. This creates a thin (~200 nm) illumination volume at the sample surface following the total internal reflection of the incident light. This enables highly selective illumination of fluorescent molecules in close proximity to the sample surface, affording high single-to-noise and low background, enabling the imaging of single molecules. This makes the techniques highly suitable for imaging events at surfaces of cells, artifical bilayers (DIBs) or other surfaces.

Timelapse of TIRF build in the lab.

By analysing molecules individually we can observe spatial and temporal organisation and gain unique insight into molecular pathways, mechanisms, rare states and intermediates that are masked by traditional ensemble measurements.

Cell imaging in collaboration with Prof Arwyn Jones, Dr Jen Wymant, Prof Mark Gumbleton & Mr Jack Sim – Cardiff University.