The intensity of the evanescent field established normal to a medium boundary in refractive index at which total internal reflection (TIR) has occurred decays steeply with distance from the boundary (as represented here by the interface between a glass coverslip and a water-based pH buffer), implying a thin excitation field for fluorophores typically ~100nm high. Bulk fluorophores are not excited and so contrast can be increased to such a high level as to make single fluorophore localization possible, even in vivo as illustrated with the bacterial cell.

There are two popular methods for achieving TIRF, objective and prism-based methods. We currently utilise the objective approach, but are implementing prism methods in the form of detachable modules, objective based (Fig2, left-panel), or prism-based (Fig2, right-panel).

Our custom-built TIRFM allows incident excitation light to be split between a TIRF path and an independent focussed laser spot ("trap") path. This permits both focussed laser photobleaching and laser-tweezing experiments, whilst simultaneously monitoring fluorescence emission from either TIRF or conventional widefield epifluorescence. Excitation is via one or more laser sources, or via mercury arc illumination. Some investigations will utilise quantitative FRET (Foerster Resonance Energy Transfer) to assess the nature of in vivo interactions between different protein components of a bacterial cell.

Our primary low-light detector is a cooled, back-thinned electron-multiplying CCD (iXon DV860, Andor), which has a QE>90% and a bandwidth of several kHz. Utilizing this camera we were able visualise a rotating fluorescent bead stuck to a bacterial filament and deduce changes in the bead position to a precision of 5nm. In doing this we were able to measure sub-steps in the bacterial motor rotation, consistent with an average of ~26 per revolution which tallied with the known copy number for the motor rotor protein FliG.